Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells

Accepted Manuscript Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells Chutima Kaewpiboon, Nawong Boonnak, Sirichat Kaowinn, Young-Hwa Chung PII: DOI: Reference:

S0960-894X(17)30774-6 http://dx.doi.org/10.1016/j.bmcl.2017.07.066 BMCL 25179

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

10 June 2017 22 July 2017 25 July 2017

Please cite this article as: Kaewpiboon, C., Boonnak, N., Kaowinn, S., Chung, Y-H., Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells, Bioorganic & Medicinal Chemistry Letters (2017), doi: http:// dx.doi.org/10.1016/j.bmcl.2017.07.066

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Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells Chutima Kaewpiboona,*, Nawong Boonnakb, Sirichat Kaowinnc, Young-Hwa Chung c,*

a

Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93210,

Thailand b

Department of Basic Science and Mathematics, Faculty of Science, Thaksin University,

Songkhla 90000, Thailand c

Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 46241,

Republic of Korea

*Corresponding authors: Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand Tel: +667460-9600 ext.2275 Fax: +667469-3992 E-mail : [email protected] (C. Kaewpiboon), Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 46241, Republic of Korea, Tel: +82515106116 Fax: +8151514-2358, E-mail : [email protected] (Y.H Chung)

Abstract Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto).The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanXinduced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The cotreatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4.

Keywords MDR cancer; formoxanthone C; apoptosis; autophagy; HDAC4

Plants have been a rich source of compounds used as therapeutic agents, and 75% of currently prescribed drugs worldwide are derived from plant sources 1. One potential source of therapeutic compounds is the Cratoxylum genus, and various species have been evaluated for biological activities of their secondary metabolite compounds, including xanthones, triterpenoids, and flavonoids 2. Formoxanthone C (XanX), a xanthone, was isolated from the green fruit of Cratoxylum formosum ssp. pruniflorum 3. This xanthone has many biological activities, including antimicrobial 4, antioxidant 5, antimalarial, and cytotoxic activities 6. Some xanthone derivatives isolated from this plant exhibit cytotoxic activity against the NCIH187 human small-cell lung cancer cell line 6. However, a molecular mechanism for anticancer activity of XanX has not been determined. Moreover, a major problem in cancer therapy is multidrug resistance (MDR) by overexpression of the drug efflux protein, Pglycoprotein (P-gp). A membrane transporter protein, P-gp is an energy-dependent drug efflux pump that maintains intracellular drug concentrations below cytotoxic levels, thereby decreasing the cytotoxic effects of a variety of chemotherapeutic agents 7-10. P-gp also plays a role in inhibition of drug accumulation and caspase activation in the MDR tumor 11, 12. Recent studies have shown that signal transducer and activator of transcription (Stat)1-histone deacetylase 4 (HDAC4)-mediated up-regulation of P-gp plays a critical role in anticancer drug resistance 13. Autophagy degrades long-lived cytoplasmic proteins and organelles, and provides nutrients in starvation or stress conditions

14

through programmed processing where

autophagy-related gene (Atg) products are sequentially involved. Autophagy is necessary for cellular homeostasis, and it is involved in biological processes including development, aging, and degeneration 15. However, aberrant regulation of autophagy is related to many diseases, such as cancer and neurodegenerative disease

16

. In particular, the first report connecting

autophagy to cancer showed that allelic loss of the essential autophagy gene Beclin1 (Becn1)

is prevalent in human breast, ovarian, and prostate cancers

17

, and that Becn1+/- mice

developed mammary gland hyperplasias, lymphomas, and lung and liver tumors

18

.

Subsequent studies demonstrated that Atg5-/- and Atg7-/- livers give rise to adenomas 19. These lines of evidence suggest that autophagy plays a role as a tumor suppressor in cancer development. Therefore, the inhibition of autophagy by pharmaceutical drugs sensitized apoptotic cell death; furthermore, co-treatment with autophagy inhibitor and chemotherapy accelerated tumor cell death compared with treatment with chemotherapy alone 20. This study was initiated to explore whether the XanX derived from the green fruit of C. formosum ssp. pruniflorum in Thailand can reverse MDR in A549RT-eto cells. We can report that XanX induces not only apoptosis but also autophagy in A549RT-eto cells and confers cytotoxicity through down-regulation of HDAC4. We therefore suggest that XanX is a very promising drug candidate for the treatment of MDR lung cancer. Formoxanthone C induces apoptosis in A549RT-eto cells A549 lung cancer cells resistant to etoposide (A549 RT-eto) exhibit enhanced P-gp protein levels that lead to MDR

13

. Thus, we selected natural compounds derived from

medicinal plants in Thailand to reverse MDR. Compound 1, isolated from C. formosum ssp. pruniflorum, was characterized as formoxanthone C. The molecular structure of XanX is shown in Figure 1A. XanX efficiently induced cell death of A549RT-eto cells in a dosedependent manner after 72 h exposure of A549RT-eto cells to XanX at two-fold serial dilutions from 25 to 1.56 µg/ml (Figure 1B). After the various concentrations of XanX were used to treat A549 parental and A549RT-eto cells for 72 h, the half-maximal inhibitory concentration (IC50) values were determined. The results showed that the IC50 value for A549 parental cells was 2.56±0.20 and that of A549RT-eto cells was 4.65±0.03, which suggested that A549 parental cells were more sensitive to growth inhibition with XanX treatment (Figure 1C). We further optimized the concentration: at a fixed time at 24 h post-treatment

and treatment time at a fixed concentration. We found that A549RT-eto cells survive in the presence of XanX at a concentration of less than 10 µg/ml, but cell death is seen at 20 µg/ml of XanX for 24 h of treatment. Therefore, we refined the dose to 20 µg/ml XanX and treated A549RT-eto cells with XanX at that concentration for various durations. We found that A549RT-eto cells are resistant to XanX at 12 h post-treatment, but eventually succumbed to XanX at 24 h post-treatment (Figure 1D). Next, we wondered whether XanX-induced cell death could be attributed to apoptosis. When A549RT-eto cells were treated with XanX (10 and 20 µg/ml) at 12 h post-treatment, we found that XanX treatment reduces expression levels of pro-caspase 8 and induces cleavage of PARP, a substrate of active caspases 3 and 7 (Figure 1E). In addition, we found that XanX treatment reduces expression levels of procaspase 9 (Figure 1E). The result indicates that XanX induces not only extrinsic but also intrinsic apoptosis.

Figure 1. Chemical structure of formoxanthone C (XanX) and the cytotoxic effect of XanX on A549RT-eto cells (A) The chemical structure and molecular weight of XanX. (B) A549RT-eto cells were treated with XanX (0, 1.56, 3.13, 6.25, 12.5, and 25 µg/ml) for 72 h. After treatment, the morphological changes of cells and cell viability were observed under a light microscope. (C) The IC50 of XanX against A549 and A549RT-eto cells was measured by MTT assay. The results shown are the average of triplicates; the bar indicates standard deviation. (**: A549 versus A549 RT-eto, p < 0.005) (D,E) A549RT-eto cells were treated with XanX (0, 5, 10, and 20 µg/ml) for 24 h after treatment and were treated with XanX (20 µg/ml) at 0, 6, 12, and 24 h. After treatment, the cell lysates from A549RT-eto cells treated with XanX were prepared and separated on a 12% SDS-PAGE gel. The expression of pre-caspase 8, precaspase 9, and cleaved-PARP proteins was detected by immunoblotting. XanX also induces autophagy in A549RT-eto cells Since it has been well known that autophagy has been implicated in the suppression of cancer development

21, 22

, we wondered whether XanX could induce autophagy in A549RT-

eto cells. Because autophagy is characterized by the formation of vacuoles, green fluorescent protein (GFP)-LC3 puncta, and conversion of LC3-I to LC3-II, we examined the autophagic features in A549RT-eto cells post-treatment with XanX. We found some vacuoles in A549RTeto cells treated with XanX (20 µg/ml) (Figure 2A). We also clearly observed accumulation of GFP-LC3-II puncta in A549RT-eto cells treated with XanX, while we did not see GFPLC3 puncta in A549RT-eto cells treated with dimethylsulfoxide (DMSO) (Figure 2B). Moreover, we found induction of LC3-I to LC3-II and overexpression of beclin-1 (Figure 2C), required for the formation of autophagic vesicles

23

. We also found a reduction in the

phospho-mTOR level, an inhibitor of autophagy after XanX treatment, indicating that XanX treatment clearly induces autophagy in A549RT-eto cells (Figure 2C).

Figure 2. Induction of autophagy by treatment with XanX in A549RT-eto cells (A) The morphology of A549RT-eto cells was observed under a light microscope at 12 h after treatment with XanX (20 µg/ml) (x100 magnification). (B) A549RT-eto cells were transfected with pEGFP-LC3B or pEGFP vector, and then treated with XanX (20 µg/ml) or DMSO as a control at 24 h post-transfection. GFP puncta were then analyzed using a fluorescence microscope at 24 h post-treatment. (C) Cell lysates from A549RT-eto cells treated with XanX (0, 7.5, 15, and 20 µg/ml) were prepared at 12 h post-treatment and separated on a 12% SDS-PAGE gel. LC3B-I cleavage, beclin-1, P-gp, phospho-mTOR, and mTOR proteins were examined by immunoblotting with the corresponding antibodies. Suppression of autophagy does not inhibit XanX-induced apoptosis, and inhibition of apoptosis does not block XanX-induced autophagy. Since recent literature has suggested that both autophagy and apoptosis are implicated in cell death in a cooperative or an independent manner

24, 25

, we wondered whether

autophagy impairment with suppression of beclin-1 hinders or accelerates XanX-induced

apoptosis. We first optimized the beclin-1 siRNA concentration (100 nM) for suppression of beclin-1 expression (data not shown). Under controlled siRNA treatment, XanX induced autophagy characterized by up-regulation of beclin-1 and LC3-I-to-LC3-II conversion levels in A549RT-eto cells (Figure 3A). Suppression of beclin-1 expression with siRNA inhibited the conversion of LC3-I to LC3-II, as expected (Figure 3B). Under this suppression of autophagic progression, XanX-induced cell death was inhibited (Figure 3A), but we still observed cleavage of PARP in beclin-1 siRNA-treated cells (Figure 3B). This result indicates that XanX-induced autophagy contributes to cell death but does not affect the XanX-induced apoptotic pathway. Conversely, when we inhibited the apoptotic pathway in A549RT-eto cells with ZVAD, a pan-caspase inhibitor, we found that Z-VAD blocks XanX-induced cell death, resulting in 85% cell survival compared with 30% cell survival with XanX treatment alone (Figure 3C). Therefore, we observed less cleavage of PARP after the combined treatment with Z-VAD and XanX compared with that following Z-VAD treatment alone (Figure 3D). Of interest, we found that Z-VAD treatment did not block conversion of LC3-I to LC3-II or beclin-1 induction (Figure 3D). This result indicates that the XanX-induced apoptotic pathway contributes to cell death and is independent of the autophagic pathway. Based on these results, we illustrated the relationship between apoptosis and autophagy during XanXinduced cell death (Figure 3E).

Figure 3. The relationship between apoptosis and autophagy during XanX treatment in A549RT-eto cells (A) A549RT-eto cells were treated with XanX (0 and 20 µg/ml) for 24 h after transfection with a control or beclin-1 siRNA (100 nM). Cell viability was observed under a light microscope and relatively measured using the MTT assay. The data were calculated as the percentage of relative cell viability and expressed as the mean of three experiments. (***: control siRNA+ DMSO versus beclin-1 siRNA+XanX, p < 0.0001) (B) Cell lysates from the treated A549RT-eto cells were prepared and separated on a 12% SDS-PAGE gel. Cleavage of PARP protein was detected by immunoblotting for apoptosis. Protein levels of beclin-1 and cleavage of LC3-I were examined by immunoblotting with the corresponding antibodies. (C) A549RT-eto cells were treated with XanX (20 µg/ml) alone, Z-VAD (20 µM) alone, and XanX plus Z-VAD for 24 h; the morphological changes of cells were observed under a light

microscope and cell viability was measured by MTT assay. The data were calculated as a percentage of relative cell viability and expressed as the mean of at least three experiments. (***: XanX versus XanX+Z-VAD, p < 0.001) (D) Cell lysates from the treated A549RT-eto cells were prepared and separated on a 12% SDS-PAGE gel. The expression of cleavage of PARP protein was detected by immunoblotting for apoptosis, and protein levels of beclin-1, LC3B, and Atg5 were compared by immunoblotting with the corresponding antibodies for autophagy. (E) Relationship between apoptosis and autophagy during XanX treatment in A549RT-eto cells. XanX induces both apoptosis and autophagy, leading to cell death. However, apoptosis does not affect autophagy and, conversely, autophagy does not affect apoptosis either (

).

Suppression of HDAC4 sensitizes XanX-induced apoptosis and autophagy in A549RTeto cells There have been recent documents showing positive roles of HDAC4 in cancer development and drug resistance

26, 27

. Our study examined expression levels of HDAC4 in

A549Rt-eto cells. We found that levels of both mRNA and HDAC4 proteins were significantly enhanced in A549RT-eto cells compared with those in A549 parental cells (Figures 4A,B). Moreover, when we examined levels of the proteins HDAC4 and P- gp during XanX treatment, we found that XanX treatment decreased expression levels of HDAC4 and P-gp in A549RT-eto cells (Figure 4C). To address the role of overexpressed HDAC4 in A549RT-eto cells, we introduced HDAC4 siRNA and examined cell viability during XanX treatment. HDAC4 siRNA treatment alone did not cause cell death in A549RTeto cells, and treatment with XanX (20 µg/ml) alone induced approximately 60% cell death at 24 h post-treatment (Figure 4D). The combined treatment with XanX and HDAC4 siRNA resulted in 80% cell death, indicating that suppression of HDAC4 accelerates XanX-mediated cell death in A549RT-eto cells (Figure 4D). This result was confirmed by an increase in

cleaved PARP fragments, conversion of LC3-I to LC3-II, and induction of beclin-1 during the combined treatment with XanX and HDAC4 siRNA over those seen during co-treatment with XanX and control siRNA (Figure 4E). These results suggest that overexpression of HDAC4 contributes to survival of A549RT-eto cells against XanX-induced apoptosis and autophagy.

Figure 4. Roles of HDAC4 in XanX-induced apoptosis and autophagy (A) Total RNAs from A549 and A549RT-eto cells were isolated and subjected to RT-PCR. Transcripts of HDAC4 were examined after optimization of PCR. Relative mRNA ratio of HDAC4 was described in comparison with mRNA levels of β-actin after measurement of band intensities using Multi Gauge Version 2.1 (Fuji, Tokyo, Japan). (**: A549 versus A549RT-eto, p < 0.005) (B) Cell lysates from A549 and A549RT-eto cells were prepared and separated on an 8% SDS-PAGE gel. The protein levels of HDAC4 were compared between

A549 and A549RT-eto cells using immunoblotting. (***: A549 versus A549RT-eto, p < 0.001) (C) A549 and A549RT-eto cells were treated with XanX (20 µg/ml) at 24 h. Cell lysates from A549 and A549RT-eto cells were prepared and separated on an 8% SDS-PAGE gel. The levels of HDAC4 and P-gp proteins, and cleavage of PARP protein, were detected by immunoblotting with the corresponding antibodies. (D, E) A549RT-eto cells were then treated with XanX (20 µg/ml) for 24 h after transfection with a control or HDAC4 siRNA (100 nM). Cell viability was observed under a light microscope and measured using the MTT assay. The data were calculated as a percentage of relative cell viability and expressed as the mean of three experiments. (*: control siRNA+DMSO versus HDAC4 siRNA+XanX, p < 0.05) Cell lysates from the treated A549RT-eto cells were prepared and separated on a 12% SDS-PAGE gel. HDAC4, beclin1, and cleavage of LC3-I protein levels were detected by immunoblotting with the corresponding antibodies. Medicinal plants are important drug candidate sources for the potential development of effective anticancer agents. More than half of the today’s anticancer drugs have been originally synthesized from natural products and their derivatives

28

. In this study, we found

that a purified compound (formoxanthone C; XanX) from C. formosum ssp. pruniflorum exhibits significant anti-proliferative effects against A549 cancer cells resistant to etoposide (A549RT-eto). Since our previous study has shown that A549-RT-eto cells display drug resistance because of overexpression of P-gp encoded by the MDR1 gene 13, it is meaningful that XanX diminishes levels of P-gp, leading to the reversal of MDR in A549RT-eto cells. A previous study showing that treatment with an HDAC inhibitor decreases P-gp expression in A549RT-eto cells 13 supports the theory that XanX-induced reduction of HDAC4 levels might be ascribed to a decrease in P-gp expression, resulting in enhanced sensitivity to etoposide. Therefore, XanX may be a highly effective anti-MDR cancer drug candidate.

Although another study has shown that XanX possess in vitro cytotoxicity against a variety of cancer cell lines 4, the molecular mechanism by which this compound exerts cytotoxicity has not been revealed. We, first, provide a line of evidence demonstrating the mechanism of the activity of XanX against A549RT-eto cancer cells. In this study, we demonstrated that XanX treatment induces not only apoptosis but also autophagy. Several natural product compounds can induce both apoptosis and autophagy cell death in various MDR cancers

29-31

In addition, our previous study reported that HDAC4 is associated with

MDR in A549RT-eto cancer models 13, and this study also showed that HDAC4 is involved in cell survival against XanX-induced apoptosis and autophagy. These results, therefore, suggest that HDAC4 can be an important target for anticancer treatment. Supporting our results, other studies have shown that Psammaplin A, a natural compound from a marine sponge that inhibits HDAC, exhibits anticancer activity

32, 33

. Vorinostat (Zolinza) and depsipeptide

(Istodax) are HDAC inhibitors that have been approved by the US Food and Drug Administration (USA) for use in the treatment of T-cell lymphoma 34.

Acknowledgments This study was supported by a research fund provided from the Faculty of Science, Thaksin University, Thailand, and a BK21+ research grant funded by the Ministry of Education, Korean government. Nawong Boonnak thanks IPST for research funds for DPST graduate with first replacement (No.03/2557).

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