Kaempferol, a potential cytostatic and cure for inflammatory disorders

Accepted Manuscript Kaempferol, a potential cytostatic and cure for inflammatory disorders Peramaiyan Rajendran, Thamaraiselvan Rengarajan, Natarajan Nandakumar, Rajendran Palaniswami, Yutaka Nishigaki, Ikuo Nishigaki PII:

S0223-5234(14)00738-7

DOI:

10.1016/j.ejmech.2014.08.011

Reference:

EJMECH 7244

To appear in:

European Journal of Medicinal Chemistry

Received Date: 11 November 2013 Revised Date:

4 August 2014

Accepted Date: 4 August 2014

Please cite this article as: P. Rajendran, T. Rengarajan, N. Nandakumar, R. Palaniswami, Y. Nishigaki, I. Nishigaki, Kaempferol, a potential cytostatic and cure for inflammatory disorders, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights

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• Kaempferol acts as both a chemopreventive and chemotherapeutic. • It acts to ameliorate various disorders, importantly including cancer.

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• Kaempferol targets various key players involved in cancer development.

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Kaempferol, a potential cytostatic and cure for inflammatory disorders

Peramaiyan Rajendran,a Thamaraiselvan Rengarajan,a Natarajan Nandakumarb Rajendran

NPO-International Laboratory of Biochemistry, 1-166, Uchide, Nakagawa-ku, Nagoya 454-

0926, Japan. Phone: +81 5236 11601 b

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Palaniswamic Yutaka Nishigaki,a and Ikuo Nishigakia.

Department of Microbiology, Immunology and Genetics, Ben Gurion University of the Negev, Beer Sheva, 84105, P.O.B.653, Israel c

Department of Applied Zoology and Biotechnology, Vivekananda College, Affiliated to

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Madurai Kamaraj University, Thiruvedakam West, Madurai -625234, India.

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*To whom correspondence should be addressed. E-mail: [email protected]

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Abstract: Kaempferol

(3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one)

is

a

flavonoid found in many edible plants (e.g., tea, broccoli, cabbage, kale, beans, endive, leek,

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tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine (e.g., Ginkgo biloba, Tilia spp, Equisetum spp, Moringa oleifera, Sophora japonica and propolis).Its anti-oxidant/anti-inflammatory effects have been demonstrated in various disease models, including those for encephalomyelitis, diabetes, asthma, and

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carcinogenesis. Moreover, kaempferol act as a scavenger of free radicals and superoxide radicals as well as preserve the activity of various anti-oxidant enzymes such as catalase, glutathione peroxidase, and glutathione-S-transferase. The anticancer effect of this flavonoid is mediated

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through different modes of action, including anti-proliferation, apoptosis induction, cell-cycle arrest, generation of reactive oxygen species (ROS), and anti-metastasis/anti-angiogenesis activities. In addition, kaempferol was found to exhibit its anticancer activity through the modulation of multiple molecular targets including p53and STAT3, through the activation of caspases, and through the generation of ROS. The anti-tumor effects of kaempferol have also been investigated in tumor-bearing mice. The combination of kaempferol and conventional

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chemotherapeutic drugs produces a greater therapeutic effect than the latter, as well as reduces the toxicity of the latter. In this review, we summarize the anti-oxidant/anti-inflammatory and anticancer effects of kaempferol with a focus on its molecular targets and the possible use of this flavonoid for the treatment of inflammatory diseases and cancer.

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Abbreviations:

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Keywords: Kaempferol, Chemoprevention, Inflammatory Diseases, Flavanoids, Apoptosis.

CRP: reactive C-protein, RASFs: Rheumatoid Arthritis Synovial Fibroblasts, PG: prostaglandins, GCLC: glutamate-cysteine ligase, catalytic, ROS: Reactive oxygen Species, JNK : c-Jun N-terminal kinase, TLR: toll-like receptor,TNF:Tumor necrosis factor,IL:Interlukin, MAPKS: Mitogen-activated protein kinase,LPS: Lipopolysaccharides,STAT: Signal transducer and activator of transcription,∆PUFA: Polyunsaturated fatty acid,BALF: Bronchoalveoloar lavage fluid,IKKβ: I-κB kinase β,TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand,PARP: Poly(ADP-ribose) polymerase,HMGB1: High-mobility group box 1,PUMA: p53 upregulated modulator of apoptosis,ATM: Ataxia-telangiectasia mutated,VEGF: Vascular endothelial growth factor,AKT: Protein Kinase B.

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Introduction:

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Flavonoids are one of the largest classes of natural polyphenolic compounds, occurring in fruits and vegetables that are regularly consumed by humans. They display a host of biological properties, including anti-oxidant, anti-carcinogenic, and anti-inflammatory ones [1,2]. The association between flavonoid intake and reduced disease risk was originally theorized to be

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attributable to the antioxidant activities of these compounds. Flavonoids, however, are metabolized in vivo; and their antioxidant capacity is thereby reduced [3]. Advancements in technology have allowed scientists to identify the active components in various herbal extracts.

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For example, paclitaxel (Taxol), one of the widely used chemotherapy medicines, was obtained from the bark of the Pacific yew, Taxus brevifolia, in 1967 through a large-scale screening program conducted by the US National Cancer Institute [4]. Due to the development of treatment-related complications, such as drug resistance and adverse effects, conventional medicine is still insufficient to provide a complete treatment of certain diseases; and, as such, continuing research to discover new drugs is needed to provide alternative therapy, either to

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complement or to replace existing conventional medicine [5]. Kaempferol, myricetin, and quercetin are members of a flavonoid subclass referred to as flavonols and are found in many foods, including onions and tea [6,7]. Although the predominant component of these major

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flavonols in edible plants is quercetin, followed by myricetin and kaempferol [6,8] all 3 of them have antioxidant capacity [9]. Therefore, kaempferol has been relatively unnoticed in comparison to quercetinand myricetin; and few in vitro studies have assessed it. Recently,

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flavonoids possessing antioxidant properties have been suggested to offer new, possible strategies for cancer chemotherapy. It has been reported that kaempferol-induced activation of antioxidant enzymes may play an important role in apoptosis in human lung non-small carcinoma H460 cells [10]. In addition, many studies have reported that kaempferol significantly inhibits the growth of cancer cells in vitro [11-13] and that this inhibition is effected by a signal transduction pathway leading to apoptosis.

This flavonol triggers apoptosis in various cells,

such as leukemia cells, lung cancer cells, and glioblastoma [14,10,15] although the cellular mechanisms underlying the action of kaempferol- induced cell-cycle arrest and apoptosis are still

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unclear [16]. The focus of this review is to discuss the molecular targets modulated by kaempferoland thepotential use this drug as a therapeutic against inflammatory disorders and cancer. Anti-oxidant and anti-inflammatory effects of kaempferol

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Inflammation is a type of non-specific immune reaction that occurs in response to injury or infection. Though it is self-limiting under normal conditions, the inflammatory reaction may go uncontrolled in certain disorders, leading to continuous or chronic inflammatory diseases [17].

Numerous studies have shown that extensive oxidative stress can lead to chronic

neurological disorders [18].

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inflammation, which, in turn, results in diseases such as cancer and in cardiovascular and Chronic inflammatory conditions such as Helicobacter pylori

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infection and Hepatitis B viruses have been linked to gastric cancer and hepatocellular carcinoma, respectively [19]. Kaempferol, with its anti-oxidant and anti-inflammatory effects, has prompted many scientists to investigate its molecular mechanisms and potential use for the treatment of inflammatory diseases.

Kaempferol as a free-radical scavenger Kaempferol has been

reported to decrease the brain damage induced by

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ischemia/reperfusion in rats [20]. In a more recent study, the authors reported that the flavonoid compound kaempferol suppressed the increase in ROS levels elicited by lipopolysaccharide in RAW264.7 rat macrophages, and ameliorated the bladder hyperactivity caused by potassium chloride after potamine sulfate-induced bladder injury [21]. Also, the inhibitory effect of

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kaempferol on ROS production in the bladder tissue was confirmed. Furthermore, another experiment showed that the aforementioned inhibitory effects were mediated through inhibition

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of induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes. Cyclophosphamide-induced cystitis in rats a well-known cystitis model, has also been reported to be mediated through induction of iNOS and COX-2.Furthermore, an iNOS inhibitor or COX-2 inhibitor has been reported to ameliorate bladder hyperactivity associated with inflammation.

Kaempferol effect on autoimmune diseases

Several studies have demonstrated the potential of kaempferol in improving the condition of many different types of autoimmune disease, including diabetes, arthritis, and asthma. For 4

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example, kaempferol abrogates hyperglycemic and hypoinsulinemic responses in the streptozotocin diabetic mouse model [22,23]. It was hypothesized that these effects were mediated through the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and reactive C-protein (CRP) and through changes in the nuclear factor kappa B (NF-

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κB) pathway[24],which is involved in the transcriptional machinery for the production of inducible nitric oxide synthase and hence for that of NO. In addition, kaempferol was found to reverse both elevated serum glucose and lowered serum insulin levels in streptozotocin or streptozotocin-nicotinamide diabetic rat models [25,26].

These results indicate that the

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therapeutic effects of kaempferol on diabetes may involve the modulation of serum glucose and insulin levels. Furthermore, kaempferol was found to improve the neuropathy [27]

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andnephropathy [28,29] seen in the streptozotocin diabetic rat model, suggesting that it could improve these potential complications associated with diabetes. In addition, this flavonol was found to reduce the incidence and severity of collagen-induced arthritis in rats [30]. Another study also showed that kaempferol can significantly reduce serum nitric oxide, urea, and creatinine levels, as well as prevent kidney dysfunction, all of which are commonly observed in animal model of nephrotoxicity [31]. The effectiveness of kaempferol is also demonstrated by

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the ability of kaempferol to inhibit the proliferation of both unstimulated and IL-1β-stimulated RASFs(Rheumatoid Arthritis Synovial Fibroblasts) in vitro, as well as the mRNA and protein expression of MMP-1, MMP-3, COX-2, and PGE2 induced in these cells

by IL-1β [32]. The

same study showed that kaempferol also inhibits the phosphorylation of ERK-1/2, p38, and JNK,

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as well as the activation of NF-κB induced by IL-1β (Fig 1). These results indicate that kaempferol should inhibit synovial fibroblast proliferation in rheumatoid arthritis, as well as

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the production of MMPs(Matrix metallo proteases), COX-2, and PGE2, which are involved in articular inflammation and destruction in this disease [32]. In addition, kaempferol was found to decrease the expression of bone turnover markers including alkaline phosphatase [33, 34]. Ina mouse model of asthma, kaempferol-induced inhibition of activation of Tyk2, a nonreceptor tyrosine kinase, was observed in OVA-induced asthma in these animals [35]. Additionally, LPS stimulated the activation of STAT1/3 signaling concomitant with down-regulated expression of Tyk-inhibiting SOCS3. In contrast, kaempferol encumbered STAT1/3 signaling with restoration of SOCS3 expression. Consistently, oral administration of kaempferol blocked STAT3 transactivation

elevated

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challenge.

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that kaempferol alleviates airway inflammation through modulating the Tyk2-STAT1/3 signaling response to IL-8 in the endotoxin-exposed airway epithelium in asthmatic mice, and may thus be beneficial in treating asthma by reducing the production of inflammatory mediators.

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Kaempferol as a free-radical scavenger and modulator of anti-oxidant enzymes/glutathione

Rajbir Singh et al., (2008) [36] reported that kaempferol can act as a potent scavenger of free radicals and superoxide radicals. This finding is consistent with the report by Soucek et

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al.,(2011)[37] showing that kaempferol is a potent superoxide anion scavenger and is able to inhibit iron-dependent microsomal lipid peroxidation. These results suggest that kaempferol is a

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radical scavenger with a potential role in the prevention and treatment of oxidative stress.Antioxidant enzymes are important to neutralize free radicals or superoxide radicals generated from many different sources. These enzymes reportedly play an important role in the effect of kaempferol. For example, Shakya et al., (2013) [38] showed that the decreased expression of

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catalase, glutathione peroxidase, glutathione-S-transferase, and reduced glutathione in liver tissue in the alcohol- and ∆PUFA-induced oxidative stress rat can be reversed by kaempferol. Additionally, the diethylnitrosamine-induced decrease in the activity and mRNA expression of

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anti-oxidant enzymes such as catalase, glutathione peroxidase, and glutathione-S-transferase, can be reversed by kaempferol [39]. Kaempferol may thus be of use to reduce the adverse effects

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arising from elevated levels of free radicals in inflammatory disorders. Kaempferol was investigated for its effects on glutathione, a tripeptide that is highly utilized in drug detoxification and can prevent damage caused by ROS [31].

Kaempferol was reported to

improve experimental Anti- -Encephalitis by in vitro, as reported by Zhang et al. (2012 40). Additionally, Kim et al.,and Shirwaikar et al.,(2006;2003) [41,42] also noted that kaempferol can reverse the decrease in reduced glutathione, glutathione peroxidase, and catalase levels seen in the kidney tissue of cisplatin and gentamicin-treated rats. The effect of kaempferol on 6

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glutathione levels in the body needs to be investigated in greater detail, because such findings may explain its role in the suppression of inflammation or possible carcinogenesis.

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Effect of kaempferol on pro-inflammatory transcription factors

NF-kB is a transcription factor that can be induced by a wide variety of stimuli, including stress, bacteria, viruses, cytokines, and free radicals. This transcription factor regulates the

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expression of many genes, including enzymes, cytokines, cell-cycle regulatory molecules, and

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angiogenic factors [43]. Chen et al., (2012) [44] reported that kaempferol can inhibit the overproduction of proinflammatory cytokines in BALF, including TNF-α, IL-1β and IL-6, and can strongly reduce the activation of MAPKs and NF-κB signaling pathways stimulated by LPS in LPS-induced acute lung injury in mice. These findings are supported by another study showing that a phenolic-rich fraction of Rhus verniciflua Stokes (Chinese lacquer tree)

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suppress TNF- and inflammatory stimulus-induced NF-kB activation [45]. The inhibition of the translocation of NF-kB into the nucleus in LPS-stimulated macrophages by kaempferol was also noted by Wall et al. (2013)[46]. In addition, Oh et al. (2011) [47] found that an extract of

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Euonymus alatus (EEA) abrogates the LPS-induced NF-κB signaling pathway by targeting

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IKKβ in RAW 264.7 cells. The beneficial effects resulting from the modulation of NF-kB by kaempferol have also been studied in several inflammatory disease models. It had been previously shown that Japanese encephalomyelitis could be prevented or ameliorated by treatmentwith kaempferol, possibly through the inhibition of NF-kB [48].

A recent study has

demonstrated the protective effects of kaempferol against rheumatoid arthritis, possibly through the inhibition of NF-kB-p65, p38, and ERK1/2 phosphorylation[32].

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Together, it is likely that suppression of NF-kB by kaempferol plays an important part in its anti-inflammatory activity. STAT3 regulates the transcription of genes involved in cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune responses [49-53].

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STAT3 is often constitutively activated in various carcinomas and interferes at different levels of tumorigenesis [50]. Siegelin et al. (2008)[54] provided evidence that TRAIL-induced apoptosis is partially driven by a kaempferol-mediated reduction in survivin protein levels. Inhibition of

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proteasomal degradation with MG132 in kaempferol-treated cells restores survivin protein levels in two glial cell lines. Consequently, over expression of survivin attenuates TRAIL-kaempferol-

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induced apoptosis. In addition, showed that kaempferol mediates down-regulation of phosphorylated Akt, thereby further reducing the survivin protein level. Furthermore, the blockage of serine/threonine kinase Akt activity by kaempferol is important for inhibition of survivin, because active phosphorylated Akt enhances the stability of survivin. However, that the

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combined treatment of cells with TRAIL and kaempferol induces cleavage (activation) of caspase-8, thereby exerting a proapoptotic effect independent of survivin, which is known not to inhibit caspase-8 activation. Other effects induced by kaempferol are a concentration-dependent

are

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suppression of X-linked inhibitor of apoptosis proteins such as Bcl-2, Bcl-xL, and Mcl-1, which members of the antiapoptotic Bcl-2 family. IL-8 production of LPS-stimulated human

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pulmonary epithelial cells could be significantly reduced by Kaempferol [55]. In this study indicated kaempferol have PARP-1-inhibiting activity in addition to the earlier described antioxidant effects. PARP-1 inhibition and preservation of cellular NAD1 and energy production could play a role in the anti-inflammatory activity of kaempferol [55].

Effect of kaempferol on inflammatory cytokines/growth mediators

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Inflammatory cytokines and mediators are key components in the inflammatory process [56].The inhibition of these targets is therefore exploited to prevent inflammation and reduce its damage.

Kaempferol significantly reduces the LPS-induced secretion of proinflammatory

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cytokines (IL-6 and IL-8) and prostaglandins (PGE2andPGF2α) in fetal membranes; IL-1βinduced COX-2 gene expression and prostaglandin production in the myometrium; and IL-1βinduced MMP-9 activity in amnion and myometrial cells [46]. It also decreases IL-1β-induced

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NF-κB p65 DNA binding activity and nuclear c-Jun expression [46]. Gong et al., (2013) [35] also found the Tyk-STAT signalling was blocked by kaempferol in both LPS-stimulated BEAS-

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2B cells and OVA-challenged mice. LPS activated Tyk2, which is responsible for the induction of eotaxin-1; whereas kaempferol dose-dependently inhibited LPS- or IL-8- induced Tyk2 activation. Similar inhibition of Tyk2 activation by kaempferol was observed in OVA-treated mice. Another study showed that kaempferol inhibits NO production and iNOS protein

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expression in Prevotella intermedia LPS-stimulated RAW264.7 cells at the translational level via HO-1-mediated ROS reduction and could be an efficient modulator of the host response in the treatment of periodontal disease [57]. Kim et al. (2012)[58] also found that kaempferol potently

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inhibits the release of HMGB1 by LPS and inhibits LPS- or HMGB1-mediated barrier permeability and expression of cell-adhesion molecules. Further studies revealed that kaempferol

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inhibits the cell-surface receptor of HMGB1and toll-like receptor (TLR) 2/4, but not the receptor for advanced glycation end products (RAGE). Collectively, these results suggest that kaempferol acts against HMGB1-mediated proinflammatory responses, thereby enrsing its usefulness as a therapeutic for vascular inflammatory diseases. Chemopreventive and anti-cancer effects of kaempferol

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Cancer has emerged as one of the top diseases in many countries, with its worldwide incidence rate increasing annually [59]. A cure for this disease is desperately needed, as the cost of treatment is not cheap and the complications from this disease invariably lead to fatal

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outcomes. A number of studies have demonstrated the anticancer effect of kaempferol isolated as an active ingredient from Nigella sativa, which favonol is active against many different types of malignancies [60]. Kaempferol has been shown to inhibit the in vitro growth of cancer cells

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from mice with benzo(a)pyrene-induced colon cancer [61] . The anti-oxidant capability of kaempferol has been implicated in the prevention of chemical-induced carcinogenesis. The

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potential of kaempferol has prompted scientists to investigate the molecular mechanism(s) involved and to evaluate the significance of this compound in the treatment of cancer. Numerous in vitro and in vivo studies have provided ample evidence that kaempferol can prevent carcinogenesis and inhibit tumorigenesis through different molecular mechanisms (Fig 2). The

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different modes of the anticancer action of kaempferol are briefly described below: Anti-proliferative effect of kaempferol

Targeting hyper-proliferative cancerous cells has been a well-established strategy in In this respect, kaempferol has been shown to be helpful for the

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cancer treatment [62].

identification of potential transcription factors that regulate differential gene expression of

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human HaCaT keratinocytes [63]. This compound was later found to inhibit the growth of many different kinds of carcinoma, including glioma/glioblastoma (LN229, U87MG, and T98G) [64,65],

breast adenocarcinoma (multi-drug-resistant MCF-7,MDA-MB-231, and BT-

549)[66,67],

leukemia (HL-60 and Jurkat)[68-70], lung cancer (H460 and A549)[71,15],

colorectal carcinoma (Caco-2, HCT-116, DLD-1, and Lovo)[72,61,73,74], pancreatic cancer (MIA PaCa-2, Panc 1)[75], osteosarcoma (U-2 OS)[76,77] and prostate cancer (PC-3,LNCaP,

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and DU145) [78-80). Kaempferol showed little effect on non-cancerous cells such as mouse fibroblasts (L929) [81] and prostate epithelial cells (BPH-1)[82]. These results suggest that kaempferol may have benefits in the treatment of different types of malignancy, while having a

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limited effect on normal human cells.

Kaempferol-mediated cell-cycle arrestin cancer cells

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Numerous studies have demonstrated the promising effect(s) of kaempferol in arresting cell- cycle progression in different types of tumor cells. Xu et al.(2008)[83]showed that

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kaempferol has a significant anti-tumor effect attributable to cell-cycle arrest and induction of apoptosis in HeLa cells, which findings suggest that kaempferol might have therapeutic potential against carcinoma of the cervix. In that study, (Xu et al., 2008) [83] kaempferol induced G2/M phase growth arrest correlated with a decrease in cyclin B1 and Cdk1 that occurred in a p53-

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independent manner; and it also caused an increase in apoptosis, which was confirmed by characteristic morphological changes, evident DNA fragmentation, and an increased apoptotic sub-G1 population. Furthermore, inhibition of NF-κB nuclear translocation, up-regulation of

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Bax, and down-regulation of Bcl-2were observed in HeLa cells treated with kaempferol, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. A recent

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study has shown that kaempferol can induce G2/M arrest in MCF-7/DOX doxorubicin-resistant breast squamous cancer cells, concomitant with increased expression of p53 and p21 proteins[84,85]. Together, these results indicate that cell- cycle arrest is likely to be one of the important mechanisms for the anticancer activity of kaempferol. Kaempferol has pro-apoptotic effects on cancer cells

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The ability of a chemotherapeutic drug to induce apoptosis is an important factor in determining its effectiveness in cancer treatment [86]. Wei Li et al. (2009)[87],using the human HCT116 colon cancer cell line, found that kaempferol induces p53-dependent growth inhibition

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and apoptosis. Kaempferol was found to induce cytochrome c release from mitochondria and to activate caspase-3 cleavage. The Bcl-2 family proteins including PUMA were involved in this process. Kaempferol also induced the phosphorylation of ATM and H2AX in HCT116 cells, and

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inhibition of ATM by a chemical inhibitor resulted in abrogation of the downstream apoptotic Using A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines [88]

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investigated kaempferol’s ability to induce apoptosis. Kaempferol inhibited cell proliferation by all three cell lines but did not cause necrosis in any of them. With respect to apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction was diminished by pre-treatment with

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a caspase-9 inhibitor, indicating the intrinsic apoptosis pathway. Western blot analysis revealed that the protein level of Bcl-xL was decreased in ovarian cancer cells, whilst p53, Bad, and Bax protein levels were up-regulated by kaempferol treatment. Another study found that kaempferol

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and TRAIL in combination drastically induced apoptosis in human colon cancer SW480 cells compared with the apoptosis obtained with either agent used singly[73]. Kaempferol markedly

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up-regulated the expression of TRAIL receptors DR5 and DR4. DR5 but not DR4 siRNA efficiently blocked apoptosis induced by the co-treatment with kaempferol and TRAIL, indicating that DR5 up-regulation by kaempferol helped to enhance TRAIL actions. Moreover, the combined effect on normal human cells was also examined. The co-treatment induced no apoptosis in normal human peripheral blood mononuclear cells and little apoptosis in normal human hepatocytes. Kaempferol induces apoptosis in ovarian cancer cells through regulating the

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expression of pro-apoptotic and anti-apoptotic proteins in the intrinsic apoptosis pathway. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating the involvement of the intrinsic apoptosis pathway. The protein level of Bcl-x(L) was decreased in

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ovarian cancer cells; whereas p53, Bad, and Bax protein levels were up-regulated by kaempferol treatment[86]. Marfe et al., (2009)[90] describes Akt inactivation and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and

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SIRT3, decrease of Bcl2, release of cytochrome c, caspase 3 activation and cell death. Sung et al., (2007) [91] also studied kaempferol to inhibit cyclin-dependent kinase 6, down-relulating

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NF-kB regulating cell proliferation, antiapoptotic and metastatic gene products. Investigating apoptosis, Kang et al. (2009)[92] detected the accumulation of a sub-G1 population of apoptotic cells, as well as the appearance of 4′-6-diamidino-2-phenylindole (DAPI)-stained apoptotic nuclei in MCF-7 cells after the administration of kaempferol. They

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showed the cleavage of Poly (ADP-ribose) polymerase (PARP), caspase-7, Bax, and caspase-9, indicating that the intrinsic pathway of apoptosis was involved. Kaempferol also down-regulated the expression of polo-like kinase 1 (PLK-1), which has been reported to regulate mitotic

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progression and to be up-regulated in several human tumors. Taken together, these findings indicate that kaempferol induces apoptosis by initiating the intrinsic caspase cascade and down-

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regulating PLK-1 expression. Altogether, these findings highlight the ability of kaempferol to induce apoptosis in cancer cells through the modulation of molecular targets and pathways. These targets can be readily exploited for therapeutic use as kaempferol rapidly moves into the clinical-trial stage.

Kaempferol can act in conjunction with chemotherapeutic drugs

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The combination of kaempferol and a chemotherapeutic agent can result in a cytotoxic effect greater than that achieved with either alone, as was shown by Luo et al. (2010)[93] who demonstrated this phenomenon in the case of the combination of kaempferoland cisplatin used

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on OVCAR-3 ovarian cancer cells. Kaempferol enhanced the effect of cisplatin through downregulation of cMyc. Kaempferol markedly increased the sensitivity of multidrug-resistant human cervical carcinoma KB-V1 cells (high Pgp expression) to vinblastine and paclitaxel dose-

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dependently, thus decreasing the relative resistance of these cells to these anti-cancer drugs [94]. None of the flavonoids tested had a significant effect on vinblastine and paclitaxel cytotoxicity in

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wild-type drug-sensitive KB-3-1 cells (lacking Pgp). Kaempferol also caused an increase in the intracellular accumulation and a reduction in the efflux, of Rh123 and 3[H]vinblastine in KB-V1 cells, but not in KB-3-1 cells. Kaempferol increased the inhibitory effectiveness of Pgp activity in MDR KB-V1 cells. Only treatment with kaempferol up to 48 h was able to significantly

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decrease the Pgp expression in a dose-dependent manner in KB-V1 cells [94]. In vivo chemopreventive and chemotherapeutic effects of kaempferol Animal models not only mirror the human body systems for evaluating the efficacy of a drug

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candidate, but also provide evidence concerning safety issues and the dosing regimen of the test drug. The anti-tumor activity of kaempferol was investigatedin terms of the effect of kaempferol

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on tissue lipid peroxidation and antioxidant status in 1,2-dimethyl hydrazine-induced colorectal cancer in male Wistar rats, and its efficacy was compared with that of irinotecan[95]. Kaempferol was shown to lower the levels of thiobarbituric acid-reactive substances in lysates of erythrocytes and liver prepared from the dimethyl hydrazine-treated rats and to rejuvenate antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. The recovery of enzyme status was maximum at the dose of 200 mg/kg body weight and was

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comparable to that affected by irinotecan, thus suggesting that kaempferol could be safely used as a chemopreventive agent in colorectal cancer. The anti-cancer effects of kaempferol in vivowere evaluated in BALB/cnu/nu mice inoculated with U-2 OS cells, and the results indicated

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the inhibition of tumor growth [96]. Barve et al. (2009) [97] reported on the oral bioavailability and pharmacokinetics of the chemopreventive kaempferol in rats. Their results suggested that kaempferol not only can exerta potent anti-tumor effect, but can also potentiate the therapeutic

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efficacy of commonly employed chemotherapeutic agents for cancer treatment.

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Kaempferol action toward cancer metastasis and angiogenesis

A number of studies have reported the effect of kaempferol on cancer metastasis and angiogenesis. The flavonol has been reported to modulate a number of key elements in cellular signal transduction pathways linked to apoptosis, angiogenesis, inflammation, and metastasis.

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Significantly, kaempferol inhibits cancer cell growth and angiogenesis and induces cancer cell apoptosis; but, on the other hand, it appears to preserve normal cell viability, in some cases even exerting a protective effect. The aim of this part of the present review was to synthesize

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information concerning the extraction of kaempferol, as well as to provide insights into the molecular basis of its potential chemo-preventive activities[98].Kaempferol mildly inhibits cell

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viability but significantly reduces VEGF gene expressionat both mRNA and protein levels in OVCAR-3 and A2780/CP70 ovarian cancer cell lines. In OVCAR-3 cells implanted on chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits angiogenesis and tumor growth of these cells. HIF-1alpha, a regulator of VEGF, is down-regulated by kaempferol treatment of both ovarian cancer cell lines. Kaempferol also represses AKT phosphorylation dose dependently. ESRRA is an HIF-independent VEGF regulator, and it is also

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down-regulated by kaempferol in a dose-dependent manner. Kaempferol has low cytotoxicity but inhibits angiogenesis and VEGF expression in human ovarian cancer cells through both HIFdependent (Akt/HIF) and HIF-independent (ESRRA) pathways and deserves further studies for

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possible application in angio prevention and treatment of ovarian cancers [99]. Lin et al. (2013)[100] demonstrated Kaempferol to inhibitAP-1-mediated MMP-2 expression and to suppress invasion and migration by oral cancer cells. Kaempferol showed significant inhibitory

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effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, attenuated inducible nitric oxide synthase, the protein

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expression of phosphorylated extracellular regulated protein kinases and metalloproteinase 9 enzyme activity. The combination of lipopolysaccharide and 12-o-tetradecanoylphorbol 13acetate promotes tumoral progression of C6 rat glioma cells via activation of metalloproteinase 9 enzyme activity and expression of the inducible nitric oxide synthase gene, which is located

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downstream of mitogen-activated protein kinase action in these cells. Kaempferol and wogonin exhibit effective inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetateinduced

events

[101].

MAPK

et

signaling

al.(2013)[76] pathways

showed

including

those

that

involving

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kaempferol attenuates

Chen

ERK, JNK, and p38, which attenuation results in the decreased DNA-binding ability of AP-1,

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and hence, down-regulation of the expression and enzymatic activities of MMP-2, MMP-9, and uPA; and thus this action contributes to the inhibition of metastasis of U-2 OS cells. Glycosylated-kaempferol Evidence suggests that some kaempferol glycosides and several kaempferol-containing

plants have antidiabetic activity and may prevent diabetic complications [102]. For instance, studies

with

kaempferol-3,7-O-α-

dirhamnoside

(kaempferitrin)

and

kaempferol

3-

16

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neohesperidoside, isolated from Cyathea phalerata stems, showed a significant hypoglycemic effect in diabetic rats [103]. This antidiabetic effect may be mediated by stimulation of glycogen synthesis, and Cazarolli et al. found that the phosphatidylinositol 3-kinase (PI3K)-glycogen

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synthase kinase 3 (GSK-3) pathway and the MAPK-protein phosphatase 1 (PP1) pathway were involved in the stimulatory effect of kaempferol 3-neohesperidoside on glycogen synthesis in rat soleus muscle [104]. This flavonoid was also found to induce a stimulatory effect on glucose

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uptake when the rat soleus muscle was incubated with very low concentrations of this kaempferol glycoside (1 and 100 nM). The authors have also showed that 100 mg/kg of

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kaempferol 3-neohesperidoside administered by oral gavage was able to increase glycogen content in the muscle, and suggested that this flavonoid stimulates glucose uptake in the rat soleus muscle via the PI3K and PKC pathways [105]. The oral administration of kaempferitrin has also been found to induce a significant hypoglycemic effect in normal and in alloxan-induced

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diabetic rats [106]. A single oral administration of two extract from the aerial parts of Equisetum myriochaetum also reduced blood glucose levels in diabetic rats; the authors proposed that kaempferol-3- sophoroside-4’-O-β-D-glucoside was responsible for this activity [107]. The

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hypoglycemic effect of a water extract from the aerial parts of Equisetum myriochaetum was later evaluated in eleven type 2 diabetic patients. The authors found that the oral administration

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of the extract (0.33 g/kg) significantly reduced blood glucose levels in these diabetic patients without significantly affecting insulin levels [108]. Fang et al. observed that kaempferol improved insulin-stimulated glucose uptake in mature adipocytes and suggested that this flavonoid could potentially act on multiple targets to ameliorate hyperglycemia, including the peroxisome proliferator-agonist receptor γ (PPAR γ) [109]. Some in vitro and in vivo studies suggest that kaempferol, some glycosides of kaempferol and plants containing this flavonoid

17

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may have neuroprotective activity and play a protective role in the development of Alzheimer's disease, Parkinson's disease or Huntington's disease [110,111]. For instance, Lopez-Sanchez et al. [110] carried out an in vivo study to evaluate the neuroprotective effect of kaempferol and

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found that the intravenous administration of this dietary agent decreased ischemia-induced brain damage in rats. This protective effect was associated with the capacity of kaempferol to reduce metalloproteinase activation, to prevent protein nitrotyrosines accumulation and to protect

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against apoptotic cell death caused by oxidative stress. Because oxidative stress is known to play an important role in some neurodegenerative disorders [112] it makes sense to think that the

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antioxidant activity of kaempferol participates in its neuroprotective activity [113]. Other mechanisms, such as reduction in amyloid β protein, may participate in the possible protective effect of kaempferol against Alzheimer's disease [114].Several reports suggest that kaempferol, glycosides of kaemperol such as tiliroside and several kaempferolcontaining plants (e.g. Tilia

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species) have anxiolytic activity in vivo [115]. In vitro and in vivo studies have also reported that kaempferol have antiallergic and antiasthmatic activity [116-120], which may be mediated, at least in part, by inhibition of histamine release. Analgesic activity has also been observed with

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[121,122].

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several kaempferol glycosides and kaempferol-containing plants in numerous in vivo studies

Kaempferol has shown other activities that may be relevant to cancer chemoprevention. The ribosomal S6 kinase 2 (RSK2), a member of the p90 (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation is known to be involved in proliferation and cell transformation induced

18

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by carcinogens. Kaempferol 3-O-(3`,4`-di-O-acetyl- α-L-rhamnopyranoside), also known as compound SL0101, was identified as the first specific inhibitor of this kinase (IC50 = 89 nM) and was also found to suppress proliferation of MCF-7 breast cancer cells [123]. The related

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compound 3Ac-SL0101 also inhibited this kinase in LNCaP prostate cancer cells [124]. This activity has also been shown by it’s a glycone kaempferol [125, 126]. Shan and O'Doherty (127) have to synthesize six SL0101 carbasugar glycoside analogues in either enantiomeric form. The

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formation of the key glycosidic bond features a highly regio- and stereospecific Pd-catalyzed cyclitolization. The functionalities on the sugar moieties have been established via corresponding

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postcyclitolization transformations. Mrozowski et al and Shan et al., [128, 129] have been shown SL0101 is a selective inhibitor of Ser/Thr protein kinases, p90 ribosomal S6 kinase (RSK). They designed a set of analogues based on the crystallographic model of SL0101 in complex with the RSK2 N-terminal kinase domain. They have identified an analogue with a 5″-n-propyl group on

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the rhamnose that has >40-fold improved affinity for RSK relative to SL0101 in an in vitro kinase assay. This analogue preferentially inhibited the proliferation of the human breast cancer line, MCF-7, versus the normal untransformed breast line, MCF-10A, which is consistent with

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results using SL0101. Kaempferol can also act as a weak estrogen receptor agonist and may cause estrogenic or anti estrogenic effects mainly depending on the concentration of endogenous

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estrogens. The anti estrogenic (and weak estrogenic) activity of kaempferol may result in inhibition of the growth of hormone-dependent cancers such as breast and prostate cancers [130133]. Kaempferol may also induce differentiation in colon cancer cells by reestablishing gap junctional intercellular communications, which are commonly impaired in cancer [134].

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Effect of kaempferol on drug-related toxicity Luo et al., (2009)[99] found that kaempferol enhances the apoptosis-promoting effect of cisplatin on ovarian cancer cells through down-regulation of cMyc

Kaempferol was found to

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potentiate the toxic effect of the chemotherapeutic agent doxorubicin by amplifying ROS toxicity and decreasing the efflux of doxorubicin[135]. Because the toxic effect of both kaempferol and doxorubicin was amplified when used in combination, this study raises the possibility of

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combinatorial therapy whose basis constitutes enhancing redox perturbation as a strategy to kill glioma cells. Organ of Corti 1 (HEI-OC1) cells were treated with kaempferol in the presence or

dose-dependent manner.

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absence of cisplatin [136].Kaempferol protected the cells against cisplatin-induced apoptosis in a Kaempferol-induced HO-1 expression protected against cell death

through the c-Jun N-terminal kinase (JNK) pathway and by the aid of Nrf2 translocation. Kaempferol alsotime-dependently increased the cellular level of GSH and the expression of

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GCLC(glutamate-cysteine ligase, catalytic).siRNA for GCLC blocked this increase in the GSH level and the protective effect of kaempferol against cisplatin-induced cell death. The expression of HO-1 by kaempferol inhibits cisplatin-induced apoptosis in HEI-OC1 cells, and the

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mechanism of protective effect is also associated with its inductive effect of GCLC expression [136]. Piao et al. (2008)[137] studied rats given both tamoxiferin and kaempferol by the oral tamoxifen due to the

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route and showed that the enhanced bioavailability of

oral kaempferol could have been due to an inhibition of CYP3A and P-gp by kaempferol. The presence of kaempferol did not alter the pharmacokinetic parameters of 4-hydroxytamoxifen, a metabolite of tamoxifen. In brief, the numerous reports on the protective effect of kaempferol against drug toxicity suggest a possible complementary role for kaempferol in improving the quality of life of cancer patients. Conclusions: 20

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Kaempferol has demonstrated its therapeutic effects against cancer and inflammation through different modes of action. This compound was found to be a potent scavenger of free radicals and superoxide radicals, while preserving the activity of various antioxidant enzymes

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such as catalase, glutathione peroxidase, and glutathione-S-transferase. These effects were shown to be beneficial in various disease models in animals, including those for experimental allergic encephalomyelitis, diabetes, asthma, and carcinogenesis. Numerous lines of evidence

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have indicated different modes of anticancer action, including anti-proliferation, cell-cycle arrest, induction of apoptosis, synergism with conventional medicines, ROS generation, and

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suppression of cancer metastasis and angiogenesis. Moreover, kaempferol can attenuate toxicity associated with the use of conventional medicines without compromising their therapeutic efficacy. Various molecular targets of kaempferol have been identified by the use of cancer cell lines; however, further research using animal models of disease is warranted to obtain more

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conclusive evidence for the molecular basis of kaempferol action. Additionally, novel kaempferol analogs/nanoparticles have been synthesized and found to possess greater anticancer and antioxidant activities than kaempferol itself. Despite its demonstrated therapeutic efficacy in

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tumor cell lines and animal models, there have been few clinical studies on kaempferol to date. More studies must be performed systematically before kaempferol can be developed into a drug

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for the potential treatment of various carcinomas and inflammatory disorders.

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