Palmatine hydrochloride mediated photodynamic inactivation of breast cancer MCF-7 cells: Effectiveness and mechanism of action

Photodiagnosis and Photodynamic Therapy 15 (2016) 133–138

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Palmatine hydrochloride mediated photodynamic inactivation of breast cancer MCF-7 cells: Effectiveness and mechanism of action Juan Wu a,b , Qicai Xiao a , Na Zhang b , Changhu Xue b , Albert Wingnang Leung a , Hongwei Zhang a , Qing-Juan Tang b,∗∗ , Chuanshan Xu a,∗ a b

School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China Laboratory of Food Science and Human Health, College of Food Science and Engineering, Ocean University of China, Qingdao, China

a r t i c l e

i n f o

Article history: Received 24 May 2016 Received in revised form 6 July 2016 Accepted 15 July 2016 Available online 18 July 2016 Keywords: Palmatine hydrochloride Photodynamic therapy Breast cancer Subcellular localization Reactive oxygen species Cell apoptosis

a b s t r a c t Breast cancer is one of the commonest malignant tumors threatening to women. The present study aims to investigate the effect of photodynamic action of palmatine hydrochloride (PaH), a naturally occurring photosensitizer isolated from traditional Chinese medicine (TCM), on apoptosis of breast cancer cells. Firstly, cellular uptake of PaH in MCF-7 cells was measured and the cytotoxicity of PaH itself on breast cancer MCF-7 cells was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Subcellular localization of PaH in MCF-7 cells was observed using confocal laser scanning microscopy (CLSM). For photodynamic treatment, MCF-7 cells were incubated with PaH and then irradiated by visible light (470 nm) from a LED light source. Photocytotoxicity was investigated 24 h after photodynamic treatment using MTT assay. Cell apoptosis was analyzed 18 h after photodynamic treatment using flow cytometry with Annexin V/PI staining. Nuclear was stained using Hoechst 33342 and observed under a fluorescence microscope. Intracellular production of reactive oxygen species (ROS) was studied by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF) using a flow cytometry. Results showed that PaH treatment alone had no or minimum cytotoxicity to MCF-7 cells after incubation for 24 h in the dark. After incubation for 40 min, the cellular uptake of PaH reached to the maximum, and PaH mainly located in mitochondria and endoplasmic reticulum of MCF-7 cells. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on MCF-7 cells, induced remarkable cell apoptosis and significantly increased intracellular ROS level. Our findings demonstrated that PaH as a naturally occurring photosensitizer induced cell apoptosis and significantly killed MCF-7 cells. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Cancer is one of the most devastating diseases all over the world. Breast cancer is one of the most common cancers in women, and its incidence rate of each year increases gradually. Conventional treatments for breast cancer mainly include chemotherapy, radiotherapy, surgery and endocrine therapy. However, these conventional treatments have some side-effect [1,2]. Therefore, novel therapeutic techniques with significantly improved outcomes are urgently needed.

∗ Corresponding author. ∗∗ Corresponding author at: College of Food Science and Engineering, Ocean University of China, No 5, Yushan Road, Qingdao, Shandong Province, 266003, PR China. E-mail addresses: [email protected] (Q.-J. Tang), [email protected] (C. Xu). http://dx.doi.org/10.1016/j.pdpdt.2016.07.006 1572-1000/© 2016 Elsevier B.V. All rights reserved.

Photodynamic therapy (PDT) is an effective and increasingly adopted therapeutic modality in the management of malignant tumors [3]. Emerging evidences have shown that PDT can directly destroy breast cancer cells through inhibition of cell growth or downregulation of HER2 gene expression in breast cancer cells [4–7]. Compared to conventional treatments, PDT has many advantages: relatively non-invasive, simply requiring illumination of the tumor site, and inducing minimal injury to the adjacent normal tissues [8]. What’s more, PDT could be repeatedly used without detrimental consequences to the patient. Most importantly, PDT is particularly suitable to elderly patients who can not bear the conventional treatments [9]. In the process of PDT, the photosensitizer (PS) plays a crucial role through generating reactive oxygen species (ROS) including hydroxyl radicals (• OH) and singlet oxygen (1 O2 ), etc. [10], which can cause significant oxidative damage to the tumor cells. Palmatine hydrochloride (PaH) is an active compound isolated from a traditional Chinese medicine rhizomes of Fibrarurea Tinctoria Lour

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[11]. It has been widely used to treat dysentery, jaundice, hypertension, and bacterial, viral and fungal infections [11–15]. PaH is a hydrochloride of palmatine (Pa) which has been proposed as a promising DNA phototherapy drug, notably due to its ability to produce 1 O2 when binding to DNA [16,17]. In the preliminary study, we found that PaH also had photosensitive activity and could kill colon adenocarcinoma HT-29 cells. The present study aims to investigate the cell apoptosis of PaH-mediated PDT on breast cancer MCF-7 cells and its action mechanisms.

MA) for live cell microscopy measurement. The next day, cells were incubated with PaH (1 ␮M) in PBS for 40 min at 37 ◦ C. After being washed twice with PBS, cells were co-loaded with 2.5 nM Mito-Tracker Green, Lyso-Tracker Green, ER-Tracker Green, and Golgi-Tracker Green (Life technologies, USA) respectively, the well-established fluorescent probes for mitochondria, lysosome, endoplasmic reticulum, and golgi apparatus respectively, and then observed under a confocal laser scanning microscopy (CLSM) (Zeiss model 510 confocal laser scanning microscope).

2. Materials and methods

2.6. Photocytotoxicity assay

2.1. Photosensitizer (PS) and light source

MCF-7 cells (1 × 104 cells/well) were seeded in 96-well plates and placed in an incubator at 37 ◦ C for 24 h. The medium was then replaced with medium containing 0, 0.04, 0.2, 1, 5, 25 ␮M PaH. After incubation for 40 min, the PaH-containing medium was washed away with PBS and replaced with fresh medium. Then the cells were irradiated with 470 nm LED light source at the energy density of 3.6 and 10.8 J/cm2 respectively. Cell viability was determined using the MTT assay [21] after incubation for 24 h at 37 ◦ C in the dark. The absorbance at 570 nm was measured using microplate reader. Three technical replicates were performed and the experiment was repeated for three times.

Palmatine hydrochloride (PaH) (HPLC ≥ 98%, CAS#: 1060502-4) purchased from Chengdu Must Bio-technology Co., Ltd, (Chengdu, China) was prepared as a stock solution of PaH (20 mM) in dimethyl sulfoxide (DMSO) and kept in the dark at −20 ◦ C until use. The light absorption spectrum of PaH has been reported in our recent report [28]. The light source consisted of a light-emitting diode (LED) array was used as described in our previous reports [18–20]. The cells for PDT treatment in the present study were exposed to blue light from the LED source with the full-width half maximum (FWHM) of 470 ± 10 nm and optical power density of 0.06 W/cm2 which was measured on the surface of the cell cultures. 2.2. Cell culture Breast cancer MCF-7 cells (ATCC, Rockville, MD, USA) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 U/mL and streptomycin 100 lg/mL; Invitrogen Co., Carlsbad, CA, USA). Cells were maintained at 37 ◦ C in a water-saturated atmosphere with 5% CO2 /95% air. 2.3. Cytotoxicity of PaH itself on MCF-7 cells MCF-7 cells (1 × 104 cells/well) were seeded in 96-well plates and placed in an incubator at 37 ◦ C for 24 h. The medium was then replaced with medium containing 0, 0.04, 0.2, 1, 5, 25 ␮M PaH. The control group was added medium without PaH. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay [21] after incubation for 24 h at 37 ◦ C in the dark. The absorbance at 570 nm was measured using microplate reader (Biotek ELX-800 Absorbance Reader, USA). Six technical replicates were performed and the experiment was repeated for three times. 2.4. Cellular uptake investigation MCF-7 cells (1 × 105 cells/ml) were incubated with PaH (1 ␮M) at 37 ◦ C in 6-well culture plates (Corning Inc., New York, NY, USA) for 0, 20, 40, 60, 80, 100, 120 min. After incubation, the cells were washed with phosphate-buffered saline (PBS, pH 7.2) and harvested. Then, the cells were resuspended in 1 ml DMSO and sonicated by the ultrasonic wave for 20 min. For determining intracellular PaH, the supernatant of sonicated cells was detected with a RF-1500PC spectrophotometer (Shimadzu) on 365 nm excitation. Three technical replicates were performed for each time slot and the experiment was repeated for three times.

2.7. Flow cytometry analysis (FCM) After PaH-mediated PDT (PaH–PDT), MCF-7 cells were incubated in 35 mm × 12 mm cell culture dishes (Fisher Scientific, Pittsburgh, PA, USA) at 37 ◦ C for 18 h. And then cell apoptosis was measured using FCM with Annexin V-FITC/PI apoptosis detection kit (Beyotime, Jiangsu, China) [22–24]. Cell apoptosis was analyzed using a FCM (Beckman Coulter, Inc., CA, USA). 10,000 cells were acquired and data processed by the software FCS Express V3. 2.8. Hoechst 33342 staining The nuclear morphology of cells was observed using a fluorescent microscope with Hoechst 33342 staining. After PaH–PDT, MCF-7 cells were incubated in cell culture dishes at 37 ◦ C for 18 h. The cells were stained using Hoechst 33342 (10 ␮g/mL) for 15 min at 37 ◦ C, then washed twice with PBS, and observed immediately under a fluorescence microscope with a filter set of Ex/Em of BP330-380/LP420 nm. The images were recorded by a colorful charge-coupled device camera. 2.9. Intracellular ROS detection Intracellular ROS was detected by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF) [25]. MCF-7 cells (1 × 105 cells/ml) were seeded in cell culture dishes and placed in an incubator at 37 ◦ C for 24 h. The next day, the cells of PaH-PDT group were incubated with PaH (1 ␮M) in PBS for 20 min and then incubated with 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 ␮M, Beyotime, Jiangshu, China) for 20 min at 37 ◦ C in the dark. After washed with PBS and replaced with fresh medium, the cells were irradiated with a 470 nm LED light source at the energy density of 10.8 J/cm2 . To examine the yield of intracellular ROS, cells were harvested and analyzed by FCM (Guava PCA, Millipore, USA) after incubation for 10 min at 37 ◦ C in the dark. Data were analyzed using the software FCS Express V3 (De Novo Software, Los Angeles, CA, USA). 3. Statistical analysis

2.5. Laser scanning confocal microscopy MCF-7 cells were seeded in 6-well culture plates containing a glass coverslip-covered 15 mm cutout (MatTek, Ashland,

Statistical analysis was performed based on one way analysis of variance. The data were expressed as the mean ± standard error (SE). All statistical analyses were performed using SPSS 11.0

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targeting of the photosensitizer to multiple subcellular organelles can greatly improve the therapeutic potential of PDT. CLSM and organelle-specific probes were used to observe the subcellular localization of PaH. As shown in Fig. 3, after incubated for 40 min, the subcellular distribution of PaH fluorescence coincided with that of Mitotracker and ERtracker, indicating that PaH was located large amount both in mitochondria and endoplasmic reticulum of MCF-7 cells. Also, we observed colocalization of PaH with Lysotracker and Golgitracker from the merged images, respectively. No localization was observed in lysosomes as well as in golgi apparatus of MCF-7 cells. 4.4. Photocytotoxicity of PaH on MCF-7 cells

Fig. 1. Viability of MCF-7 cells after incubated with PaH for 24 h in the dark. c(PaH): 0, 0.04, 0.2, 1, 5, 25 ␮M. Each error bar indicates standard error (n = 3). * P < 0.05; ** P < 0.01.

Fig. 4 compares the effects of different light energy densities combined with different PaH concentrations on MCF-7 cells. It found a significant (* P < 0.05, ** P < 0.01) reduction in viability after PaH–PDT compared to the controls. The results clearly demonstrated that cell viability after PaH–PDT was significantly decreased in a drug and light-dose dependent manner (Fig. 4a), while light irradiation alone showed no obvious cytotoxicity (Fig. 4b). The half maximal inhibitory concentration (IC50 ) of PaH was 0.087 ␮M at the energy density of 10.8 J/cm2 , indicating a high tumor-killing efficacy of PaH-PDT with a low dose of drug concentration. 4.5. Flow cytometry analysis (FCM)

Fig. 2. The time course for PaH uptake in MCF-7cells. Cells were incubated with PaH (1 ␮M) for different time. At each time point, the autofluorescence of intracellular PaH in the supernatant of broken cells was measured by a RF-1500PC spectrophotometer on 365 nm excitation, and the mean fluorescence intensities were recorded. Each error bar indicates standard error (n = 3).

software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference. 4. Results

In view that FCM with Annexin V/PI staining can clearly distinguish early apoptosis from late apoptosis or necrosis [22–24], it was carried out in this present study after PaH–PDT. The results of FCM showed that PaH-PDT increased the early and late apoptotic (necrotic) rates of MCF-7 cells. Fig. 5a showed that the early apoptotic and late apoptotic (necrotic) rates without treatment were 3.36% and 0.68%, respectively. Fig. 5b showed that the early apoptotic and late apoptotic (necrotic) rates of MCF-7 cells by blue light irradiation alone, were 4.76% and 0.56%, respectively. Fig. 5c showed that the early apoptotic and late apoptotic (necrotic) rates of MCF-7 cells after PaH treatment alone were 4.64% and 0.56%, respectively. Fig. 5d showed that the early apoptotic and late apoptotic (necrotic) rates of MCF-7 cells after PaH-PDT (1 ␮M, 10.8 J/cm2 ) markedly increased to 21.16% and 9.86%, respectively. These findings demonstrated that PaH-PDT significantly increased the rate of early and late apoptosis (necrosis) of MCF-7 cells.

4.1. Cytotoxicity of PaH itself on MCF-7 cells After incubation with different concentration of PaH in the dark for 24 h, the viability of MCF-7 cells was determined using MTT assay. As shown in Fig. 1, the viability of MCF-7 cells was not significantly decreased with the concentration of PaH lower than 5 ␮M. 4.2. Cellular uptake investigation of PaH in MCF-7 cells Cellular uptake by cancer cells is an important property of photosensitizers. The cellular uptake in MCF-7 cells was monitored through intracellular fluorescence of PaH and the supernatant was detected by fluorescence spectrophotometer. Results showed that the fluorescence intensity of PaH in MCF-7 cells was changing along with the incubation time. After incubated for 40 min, the fluorescence intensity of PaH reached the peak, and then started reducing until entering to a plateau (Fig. 2). 4.3. Subcellular localization of PaH in MCF-7 cells Subcellular localization of the photosensitizer is crucial in determining the fate of cells and cell death mechanism [26,27]. Specific

4.6. Nuclear staining analysis MCF-7 cells were stained using Hoechst 33342 18 h after PaHPDT (1 ␮M, 10.8 J/cm2 ). Changes of nuclear characteristic in cells were observed under fluorescence microscopy. The results showed that the nuclei of the three control groups were stained dimly and observed a variety of karyomorphism, including round, kidney shape and oval (Fig. 6a–c). The nuclei of apoptotic cells showed an increased fluorescence and typical apoptotic bodies such as nuclear condensation and fragmentation were observed in MCF-7 cells under the PaH-PDT (1 ␮M, 10.8 J/cm2 ) (Fig. 6d). 4.7. Production of ROS in MCF-7 cells The production of ROS in cells is crucial for photodynamic activity. In this study, the level of ROS in MCF-7 cells was analyzed using FCM with DCFH-DA staining. Results of FCM showed the spectral shift of the fluorescence curves to the right after PaH-PDT (1 ␮M, 10.8 J/cm2 ), indicating significant increase of the intracellular ROS level in MCF-7 cells (Fig. 7).

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Fig. 3. Subcellular localization of PaH in MCF-7cells. Mito-Tracker Green, Lyso-Tracker Green, ER-Tracker Green, and Golgi-Tracker Green (green) was used to label mitochondria, lysosome, endoplasmic reticulum, and golgi apparatus, respectively. Blue-white shows colocalization of PaH with fluorescent probes. Scale bar: 20 ␮m.

Fig. 4. Viability of MCF-7cells after PaH-PDT. (a) The cells were incubated with different concentrations of PaH for 40 min before blue light irradiation (energy density: 3.6, 10.8 J/cm2 ). (b) The cells were only irradiated by blue light (energy density: 3.6, 10.8 J/cm2 ). Each error bar indicates standard error (n = 3). * P < 0.05; ** P < 0.01.

5. Discussion Our recent study showed that PaH, a naturally occurring photosensitizer isolated from TCM Fibrarurea Tinctoria Lour, had photodynamic killing on colon adenocarcinoma cells. In the present study, we aim to investigate whether PaH has photodynamic activity on breast cancer cells and further discussed its action mechanisms. Our results showed that PaH itself did not exert significant damage on the MCF-7 cells under the concentration of 5 ␮M. After incubated for 40 min, the fluorescence intensity of PaH reached the peak, and then started reducing probably because the PaH was partly metabolized by the MCF-7 cells. After light irradiation, the

rate of cell death increased significantly in a PaH concentrationdependent and light dose-dependent manner. The tumor-killing efficacy of PaH-PDT on MCF-7 cells was much better than that on HT-29 cells under the same concentration and light dose according to our recent study [28]. The reasons causing the difference in PDT effectiveness might be related to the type of cells and the drug sensitivity of different cells. Cell apoptosis is an important mode of cell death after PDT. Our results from FCM showed that the early apoptotic and late apoptotic (necrotic) rates increased significantly up to 21.16% and 9.86% after PaH-PDT (1 ␮M, 10.8 J/cm2 ), demonstrating that the treatment triggered the early and late apoptosis (necrosis) of MCF-7 cells. To

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Fig. 5. Apoptosis of MCF-7 cells 18 h after PaH-PDT. Cells were stained with Annexin V-FITC and PI, and cell apoptosis was analyzed using a flow cytometry. (a): control; (b): blue light irradiation alone; (c): PaH treatment alone; and (d): PaH–PDT (1 ␮M, 10.8 J/cm2 ).

Fig. 6. Nuclear characteristic of MCF-7 cells 18 h after PaH-PDT. The nuclear characteristic of MCF-7 cells was analyzed using a fluorescence microscope with Hoechst33342 staining. (a): control; (b): blue light irradiation alone; (c): PaH treatment alone; and (d): PaH–PDT (1 ␮M, 10.8 J/cm2 ).

Fig. 7. Intracellular ROS production of MCF-7cells after PaH-PDT. Reactive oxygen species (ROS) in MCF-7 cells were analyzed using flow cytometry (FCM) with DCFH-DA staining. 1: control; 2: blue light irradiation alone; 3: PaH treatment alone; and 4: photodynamic treatment with PaH (PaH-PDT) (1 ␮M, 10.8 J/cm2 ).

confirm our above results from FCM, nuclear staining was also used in this study. Classical characteristics of apoptotic cells such as cell shrinkage, nuclear fragmentation and condensation were observed in MCF-7 cells after PaH-PDT (1 ␮M, 10.8 J/cm2 ). These findings from nuclear staining further demonstrated that PaH-PDT significantly induced cell apoptosis of MCF-7 cells. Emerging evidence showed that intracellular ROS level played an important role in photodynamic damage [29]. DCFH-DA is cell permeable, and, after uptake, it is cleaved by intracellular esterases to 2 , 7 -dichlorofluorescin (DCFH), trapped within the cells, and

oxidized to the fluorescent molecule 2 , 7 -dichlorofluorescein (DCF) by ROS [30]. In this study, we found that the level of ROS in cancer cells was increased after treatment with light-activated PaH. The subcellular organelles localization of photosensitizers are considered to be important targets of photodynamic action. For example, mitochondria have received attention as drug targets for crucial regulators of apoptosis [31]. Previous studies reported that PSs localized in the mitochondria easily induced apoptotic cell death upon light irradiation [32]. Mitochondrial damage often lowers the energy production of the cells and activates the intrinsic apoptotic pathway [33]. Endoplasmic reticulum also has many general functions involving the folding of protein molecules and the transport of synthesized proteins. In the present study, we found that PaH mainly located in mitochondria and endoplasmic reticulum, but not in lysosomes and golgi apparatus of MCF-7 cells. The subcellular localization of the photosensitizer is not only affected by the phenotypes of target cells, but also greatly affected by the physiochemical properties of the photosensitizer, such as molecular weight, lipophilicity, ionic charge and partition coefficient. It is recognized that ionic lipophilic materials such as PaH predominately localize in mitochondria, while the hydrophilic materials majorly localize in lysosomes [26,34,35]. The above findings indicated that PaH-mediated photodynamic action induced a significant increase in ROS in MCF-7 cells, caused oxide damage to mitochondria and endoplasmic reticulum, subsequently resulting in cell death of MCF-7 cells. 6. Conclusions The present study demonstrated that PaH, as a naturally occurring photosensitizer isolated from TCM Fibrarurea Tinctoria Lour, showed significant photodynamic activity on breast cancer cells. It highlight that the intracellular ROS increase and apoptosis induc-

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tion are important action mechanisms of PaH-mediated PDT on breast cancer cells. Acknowledgments This work was supported by the general grant fund from Hong Kong Research Grant Committee (476912), Health and Medical Research Fund of Hong Kong (13120442), Scientific and Technological Project in Shandong Province (2015GSF115030), National Natural Science Foundation of China (U1406402), “Taishan Scholars” Special Fund to Construction Project and Changjiang Scholars and Innovation Team Development Plan Fund (IRT1188). References [1] M. Overgaard, P.S. Hansen, J. Overgaard, C. Rose, M. Andersson, F. Bach, et al., Postoperative radiotherapy in high-risk premenopausal women with breast cancer who receive adjuvant chemotherapy Danish breast cancer cooperative group 82b trial, N. Engl. J. Med. 337 (1997) 949–955. [2] P. Casolo, D. Mosca, C. Amorotti, A. Raspadori, B. Drei, B.P. Di, et al., Our experience in the surgical treatment of early breast cancer: results of a prospective study of 204 cases, Ann. Ital. Chir. 68 (1997) 626–630. [3] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, et al., Photodynamic therapy of cancer: an update, CA: Cancer J. Clin. 61 (2011) 250–281. [4] R. Hornung, M.K. Fehr, J. Monti-Frayne, B.J. Tromberg, M.W. Berns, Y. Tadir, Minimally-invasive debulking of ovarian cancer in the rat pelvis by means of photodynamic therapy using the pegylated photosensitizer PEG-m-THPC, Br. J. Cancer 81 (1999) 631–637. [5] L.Y. Xue, S.M. Chiu, N.L. Oleinick, Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4, Oncogene 20 (2001) 3420–3427. [6] N.H. Bui-Xuan, M.K. Tang, C.K. Wong, K.P. Fung, Photo-activated pheophorbide-a, an active component of Scutellaria barbata, enhances apoptosis via the suppression of ERK-mediated autophagy in the estrogen receptor-negative human breast adenocarcinoma cells MDA-MB-231, J. Ethnopharmacol. 131 (2010) 95–103. [7] A. Nowak-Stepniowska, M. Malecki, K. Wiktorska, A. Romiszewska, A. Padzik-Graczyk, Inhibition of cell growth induced by photosensitizer PP(Arg)2-mediated photodynamic therapy in human breast and prostate cell lines. Part I, Photodiagn. Photodyn. Ther 8 (2011) 39–48. [8] P.K. Selbo, A. Hogset, L. Prasmickaite, K. Berg, Photochemical internalisation: a novel drug delivery system, Tumour Biol.: J. Int. Soc. Oncodevelopmental Biol. Med. 23 (2002) 103–112. [9] S.G. Rockson, D.P. Lorenz, W.F. Cheong, Woodburn K.W. Photoangioplasty, An emerging clinical cardiovascular role for photodynamic therapy, Circulation 102 (2000) 591–596. [10] J.F. Lovell, T.W. Liu, J. Chen, G. Zheng, Activatable photosensitizers for imaging and therapy, Chem. Rev. 110 (2010) 2839–2857. [11] Y.Q. Wang, H.M. Zhang, G.C. Zhang, Studies of the interaction between palmatine hydrochloride and human serum albumin by fluorescence quenching method, J. Pharm. Biomed. Anal. 41 (2006) 1041–1046. [12] F. Wang, H.Y. Zhou, L. Cheng, G. Zhao, J. Zhou, L.Y. Fu, et al., Effects of palmatine on potassium and calcium currents in isolated rat hepatocytes, World J. Gastroenterol.: WjG. 9 (2003) 329–333. [13] B. Liu, X. Yan, S. Cao, B. Chong, Y. Lu, Studies on the interaction of palmatine hydrochloride with bovine hemoglobin, Lumin.: J. Biol. Chem. Lumin. 29 (2014) 211–218. [14] C.W. Xiao, Q.A. Ji, Q. Wei, Y. Liu, G.L. Bao, Antifungal activity of berberine hydrochloride and palmatine hydrochloride against Microsporum canis-induced dermatitis in rabbits and underlying mechanism, BMC Complement. Altern. Med. 15 (2015) 177.

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