2,3-Dihydro-3β-methoxy Withaferin-A Protects ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 2756-2760

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2,3-Dihydro-3β-methoxy Withaferin‑A Protects Normal Cells against Stress: Molecular Evidence of Its Potent Cytoprotective Activity Anupama Chaudhary, Rajkumar S. Kalra, Chuang Huang, Jay Prakash, Sunil C. Kaul,* and Renu Wadhwa* DAILAB, National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki 305-8565, Japan S Supporting Information *

ABSTRACT: 2,3-Dihydro-3β-methoxy withaferin-A (3βmWi-A) is a natural withanolide that is structurally close to withaferin-A (Wi-A), is cytotoxic to human cancer cells, and is a candidate anticancer natural compound. Using cell-based biochemical, molecular, and imaging assays, we report that Wi-A and 3βmWi-A possess contrasting activities. Whereas Wi-A caused oxidative stress to normal cells, 3βmWi-A was well tolerated at even 10-fold higher concentrations. Furthermore, it promoted survival and protected normal cells against oxidative, UV radiation, and chemical stresses. We provide molecular evidence that 3βmWi-A induces antistress and pro-survival signaling through activation of the pAkt/MAPK pathway. We demonstrate that 3βmWi-A (i) contrary to Wi-A is safe and possesses stress-relieving activity, (ii) when given subsequent to a variety of stress factors including Wi-A, protects normal cells against their toxicity, and (iii) is a vital compound that may guard normal cells against the toxicity associated with various targeted therapeutic regimes in clinical practice. 5−10 μM (Figure 1B). Consistent with the cell viability, observations on cell morphology supported that, whereas Wi-A caused stress and cell death at 0.6 μM, 3βmWi-A showed no effect at equivalent concentrations (Figure S1A,B). In longterm clonogenicity assays, Wi-A caused inhibition of colony formation at concentrations as low as 0.1 to 0.3 μM, and 3βmWi-A exerted no significant effect even at 10-fold higher concentration (Figure S1C,D), suggesting that, in contrast to Wi-A, 3βmWi-A is safe for normal human cells. In order to validate this further, presenescent [ ∼55 population doublings (PDs)] human fibroblasts were serially passaged in medium supplemented with subcytotoxic concentrations of either Wi-A (0.1 μM) or 3βmWi-A (0.5 μM). As shown in Figure 1C, we found that the cells cultured in control and 3βmWi-Asupplemented medium entered senescence at around 70−72 population doublings. However, the cells cultured in Wi-Asupplemented medium stopped dividing at 64 PDs. The data suggested that, whereas Wi-A caused growth arrest and led to premature senescence of normal cells, 3βmWi-A was well tolerated and was safe (Figure 1D). In light of the information that Wi-A causes oxidative stress to normal cells,2 we next examined the effect of 3βmWi-A. Contrary to Wi-A, which upon treatment increased the level of ROS, 3βmWi-A-treated cells were marked by reduced reactive oxygen species (ROS) levels at doses of 1.5 to 3 μM (Figure 1D and data not shown). Expression analyses of DNA damage and stress marker proteins

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ithaferin-A (Wi-A) and 2,3-dihydro-3β-methoxy withaferin-A (3βmWi-A) are natural and co-occurring withanolides present in Withania somnifera. Wi-A, a potent natural compound, has been shown to inhibit cancer cell proliferation and migration by multiple pathways including oxidative stress,1−3 inactivation of NFκB, c-MET, AKT, RAF-1, BRCA1, HSF1, and EMT signaling, and restoration of p53mediated growth arrest or apoptosis.3−12 It has been shown to promote p21WAF1 (WAF1 is wild-type activating fragment-1), BAX, and IκBα13 function and inhibit intermediate filament protein vimentin assembly.14 Furthermore, it has been shown to sensitize cancer cells to radiotherapy15 and work in a synergistic manner with chemotherapeutic drugs including sorafenib,16 oxaliplatin,17 and cisplatin.18 The anticancer potential of Wi-A has also been validated in cellular and mouse models of tumor progression and metastasis.6,12,14,19,20 On the other hand, the effect of 3βmWi-A has not been explored in normal cells or in vivo models. Of note, we earlier reported that, in contrast to Wi-A, 3βmWi-A lacks activity in cancer cells.21 In the present study, we assessed the activities of 3βmWi-A in normal human cells. We provide cellular and molecular evidence that 3βmWi-A, while being inert to cancer cells,21 protects normal cells against stress.

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RESULTS AND DISCUSSION We examined the cytotoxicity of withaferin A (Wi-A) and βmethoxy withaferin A (3βmWi-A) (Figure 1A) to human normal cells and found that, whereas Wi-A caused cytotoxicity (∼90% of cells died) at 1 μM, 3βmWi-A was well tolerated at © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 4, 2017 Published: October 18, 2017 2756

DOI: 10.1021/acs.jnatprod.7b00573 J. Nat. Prod. 2017, 80, 2756−2760

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Figure 1. Differential activities of Wi-A and 3βmWi-A. (A) Schematic structures showing ergostane base ring of Wi-A and its methoxy withanolide 3βmWi-A (position marked with red circle on a gray background). Wi-A ring moiety structure showing substitution of the β-methoxy group at position 3 in its methoxy withanolide 3βmWi-A, marked by a red circle. (B) Viability of normal human cells treated with serially increasing doses of either Wi-A or 3βmWi-A. (C) Serial passaging of presenescent normal cells in medium supplemented with subtoxic doses of Wi-A (0.1 μM) or 3βmWi-A (0.5 μM). (D) Level of reactive oxygen species (ROS) in normal cells treated with either Wi-A or 3βmWi-A. Scale bar, 100 μm. (E) Expression of CARF and GADD45α mRNA in Wi-A- and 3βmWi-A-treated normal cells; quantitation of the signals along with statistical significance (*p < 0.05, *p < 0.01, and *p < 0.001) from three independent experiments is shown.

Figure 2. 3βmWi-A modulates the antioxidant response, whereas WiA induced activation of p53/p21WAF1 and apoptosis signaling. (A) Immunoblotting showing expression levels of p53, p21WAF1, and mortalin in control-, Wi-A-, and 3βmWi-A-treated cells; quantitation from three independent experiments is shown below. (B) Immunofluorescence staining of mortalin in control-, Wi-A-, and 3βmWi-Atreated cells (scale bar, 20 μm); quantitation from three independent experiments is shown below. (C) Immunoblots showing p53 and CARF expression levels in 0.6 μM Wi-A- or 3βmWi-A-treated cells. (D) Immunoblots of Keap1 and Nrf2 expression levels in Wi-A- and 3βmWi-A-treated normal cells; quantitation of their relative expressions from three experiments is shown. (E) Immunoblots showing Bcl-2 and Bax expression levels in control-, Wi-A-, and 3βmWi-A-treated normal cells; quantitation of their relative expressions from three independent sets is shown. Statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

CARF (collaborator of ARF) and GADD45α also revealed a dose-dependent increase in their mRNA levels in cells treated with Wi-A, and not with 3βmWi-A (Figure 1D). These data corroborated the finding that Wi-A, and not 3βmWi-A, is cytotoxic to normal human cells. Cytotoxicity of Wi-A to human cancer cells has earlier been shown to be mediated by induction of oxidative stress or activation of p53/p21WAF1 by abrogation of p53-mortalin complexes.6,22 In light of this information, we examined the activity of 3βmWi-A and found that it has no effect on the expression level of either p53 or p21WAF1 (Figure 2A and data not shown), whereas, in contrast to a decrease in mortalin expression in response to treatment with Wi-A, 3βmWi-Atreated cells showed a moderate increase in its expression (Figure 2A,B). Furthermore, consistent with the data shown in Figure 1E, cells treated with Wi-A, but not with 3βmWi-A, showed an increase in CARF and p53 proteins (Figure 2C). Wi-A-induced oxidative stress has been shown to instigate antioxidant response and apoptotic signaling.9,22 We next examined the effect of 3βmWi-A on antioxidant signaling proteins Keap1 and Nrf2 (master regulators of antioxidant response) and found that in contrast to Wi-A, which caused an initial increase in expression level of both Keap1 and Nrf2 at the onset of oxidative stress (adaptive response) followed by a decrease (apoptotic response), 3βmWi-A caused a small, but significant, gradual increase in the levels of both antioxidative stress proteins (Figure 2D). The initial increase followed by a decrease in Nrf2 levels suggested a dose-dependent response (oxidative stress to apoptosis) of cells to Wi-A. In contrast to Wi-A, 3βmWi-A-treated cells showed an insignificant change even up to 1.5 μM. Similar changes were observed in Keap-1,

which regulates Nrf2 by ubiquitination and degradation. This finding was confirmed by immunostaining of Nrf2 and Keap1 proteins. Nuclear accumulation of Nrf2 was found in Wi-A- but not in 3βmWi-A-treated cells (Figure S2A). Molecular analysis of apoptotic signaling revealed a decrease in anti-apoptotic protein Bcl-2 and an increase in pro-apoptotic protein Bax in Wi-A-treated cells only (Figure 2E). Furthermore, PCR-based transcriptional analysis of a small array of proteins involved in DNA damage, cell cycle, and oxidative stress signaling revealed a higher increase/decrease in Wi-A-treated cells (Figure S2, B and C). On the basis of the above contrasting results of Wi-A and 3βmWi-A effects, we next speculated that 3βmWi-A may have cytoprotective activity and investigated by recruiting a variety of oxidative stress models to normal cells in vitro. Normal cells exposed to H2O2 (2 h) were subsequently recovered in culture medium supplemented with either Wi-A or 3βmWi-A for 2 days. As shown in Figure 3A, H2O2-stressed cells showed ∼60% survival when recovered in control medium. Of note, whereas cells kept with Wi-A-supplemented medium showed poor recovery, 3βmWi-A caused improved survival especially at higher concentrations (Figure 3A and B). In order to investigate whether the cytoprotective effect of 3βmWi-A is specific to oxidative stress or to other stresses as well, we employed a variety of stress models including UV and genotoxic drugs (doxorubicin, cisplatin, and etoposide) that elicit ROS production. Consistent with H2O2 stress, cells given 3βmWi-A treatment (1.5 or 3 μM) post-UV irradiation or treated with doxorubicin, cisplatin, or etoposide exhibited better survival (Figure 3C−F). Furthermore, we found that (i) the increase in 3βmWi-A-induced recovery was concentration2757

DOI: 10.1021/acs.jnatprod.7b00573 J. Nat. Prod. 2017, 80, 2756−2760

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Figure 4. 3βmWi-A provides cytoprotection by activating cell survival signaling. (A) Immunostaining of cells showing expression levels of phospho-p38 and phospho-p44/42 in Wi-A- or 3βmWi-A-treated cells post-H2O2 (500 μM, 2 h). Quantitation of immunofluorescence intensities is shown below the images. Scale bar, 20 μm. (B) Immunoblot showing expression levels of pAkt, β-catenin, survivin, pp38, p-p44/42, Bcl-2, and PARP-1 proteins in cells recovered in 0.3 or 0.6 μM Wi-A or 3βmWi-A treatment post-H2O2 stress, respectively. (C) Immunostaining of γH2AX, phospho-ATM, 53BP1, and PDI in cells recovered in 0.6 μM Wi-A or 3βmWi-A post-H2O2 stress; scale bar, 20 μm. (D) Viability of cells recovered in 0, 1.5, or 3 μM 3βmWiA-supplemented medium post-Wi-A (0.3 μM)-stressed cells.

Figure 3. 3βmWi-A protects normal cells against a variety of stresses. (A) Crystal violet stained cells showing their survival in response to treatment with either Wi-A or 3βmWi-A and post-UV irradiation (50 mJ/cm2). (B) Quantitation from three experiments is shown. (C−F) Viability of cells treated with Wi-A or 3βmWi-A for 48 h treated postUV (50 mJ/cm2) irradiation, -doxorubicin (1 μM, 24 h), -cisplatin (100 μM, 24 h), or -etoposide (100 μM, 24 h), respectively. (G) Viability of cells recovered in 3 μM 3βmWi-A-supplemented medium for 48 or 72 h post-UV (50 mJ/cm2) or -H2O2 (500 μM, 2 h) treatments. *p < 0.05, **p < 0.01, and ***p < 0.001.

and 53BP1 DNA damage markers (Figure 4C). 3βmWi-Atreated cells, compared to Wi-A and control, possessed fewer γH2AX and pATM stain-positive nuclei (Figure S3B). Furthermore, expression of PDI (protein disulfide isomerase), a thioredoxin family chaperone and marker of oxidative and endoplasmic reticulum (ER) stress, decreased in 3βmWi-Atreated cells (Figure 4C), suggesting that it may protect against ER stress, which warrants further investigation. Current therapeutic regimes against cancer are enormously toxic to normal cells, evoking adverse side effects, leading to death. Protection of normal cells during chemo/radiotherapy to cancer cells is highly desirable. In view of this, we hypothesized that the toxicity of Wi-A to normal cells as reported earlier1 could be limited in the presence of 3βmWi-A. We tested it by treating cells with 3βmWi-A followed by cytotoxic Wi-A exposure. As shown in Figure 4D, Wi-A-treated (0.3 μM, 24 h) cells indeed showed better recovery in 3βmWi-A-supplemented medium.

dependent (1.5 to 3.0 μM) and (ii) extended (72 h) incubation of cells in 3.0 μM 3βmWi-A-supplemented medium either postUV or -H2O2 stress led to better recovery as compared to 48 h incubation (Figure 3G). These data established the pro-survival effect of 3βmWi-A treatment and were confirmed by expression analysis of stress marker proteins by immunostaining with specific antibodies. We found that whereas H2O2- or UVinduced increase in p53 and CARF was enhanced by posttreatment with Wi-A, 3βmWi-A treatment had no effect (Figure S3A). We next investigated the molecular mechanism of 3βmWi-A pro-survival activity. In normal cells, Akt and p38/p44 MAPK signaling cascades are the key survival pathways activated during recovery of cells from stress. Inability to stimulate these pathways has been associated with radiation- or oxidative stressinduced apoptosis.23 We investigated the Akt and p38/p44 MAPK signaling axis and found that cells treated with 3βmWiA post-H2O2 or -UV retained high levels of phosphorylated p38MAPK (Thr180/Tyr182) and p44/42(Erk1/2 at Thr202/ Tyr204), two key markers of survival and proliferation (Figure 4A; data not shown). Expression analysis of proteins in cells treated either with Wi-A or 3βmWi-A at low and moderate (0.3 and 0.6 μM, respectively) concentrations affirmed enriched levels of phosphorylated Akt (Ser473), phosphorylated p38 MAPK (Thr180/Tyr182), and p44/42 (Thr202/Tyr204), while stabilized β-catenin and enriched survivin protein levels confirmed 3βmWi-A pro-survival activity in treated cells (Figure 4B). Furthermore, a decrease in Bcl-2, PARP1 (fulllength) levels post-H2O2 in Wi-A cells changed to their restored expression in 3βmWi-A-treated cells (Figure 4B). These data provided molecular evidence for the differential regulation of apoptosis and survival pathways by Wi-A and 3βmWi-A, respectively. Consistent with these data, cells treated with 3βmWi-A (compared to control and Wi-A) postoxidative stress exhibited less DNA damage as examined by γH2AX, pATM,

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CONCLUSIONS In the present report, we assessed the activities of Wi-A and 3βmWi-A to normal human cells. We demonstrate that Wi-A, and not 3βmWi-A, is cytotoxic. 3βmWi-A, in contrast to Wi-A, supported normal cell survival under a variety of stresses. Cellbased assays demonstrated that β-methoxy substitution at carbon of the Wi-A ergostane ring alters its activity. Above alkoxy substitution to Wi-A led to abrogation of its oxidative stress-inducing effect. Furthermore, it caused stress-protecting and pro-survival properties. Such antistress and pro-survival effects of 3βmWi-A were marked by modulated expression levels of oxidative, UV, and DNA-damage response markers and induced cell survival signaling (activated pAkt, p38, p44/4, and β-catenin/survivin) (Figure 5). Transcript analysis revealed upregulated expression of several markers of growth arrest and DNA damage signaling in Wi-A-treated cells; 3βmWi-A-treated cells showed their downregulation (Figure S2, B and C). Molecular mechanism(s) of such changes at the transcriptional level warrant further studies. Of note, we found that 3βmWi-A 2758

DOI: 10.1021/acs.jnatprod.7b00573 J. Nat. Prod. 2017, 80, 2756−2760

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procedure as previously described.6 For representation of stained colonies, plates were scanned on an Epson GT-9800F scanner. Experiments were carried out in triplicates, and statistical significance was calculated using Student’s t test. Reactive Oxygen Species Assay. TIG-3 cells treated with either 0.3, 0.6, or 1.5 μM Wi-A or 3βmWi-A for 48 h were used to examine levels of ROS as described previously.22 Reverse-Transcription PCR (RT-PCR). Total RNA was extracted from Wi-A- and 3βmWi-A-treated cells with the Qiagen RNAeasy kit, as previously described.21 Complementary DNA (cDNA) was prepared from total RNA (2 μg) using ThermoScript reverse transcriptase (ThermoFisher, USA) following the manufacturer’s protocol. The cDNA was further subjected to PCR amplification with settings comprising an initial 10 min denaturation step at 95 °C, followed by 30 cycles of amplification (95 °C for 45 s, 58 °C for 1 min, and 72 °C for 45 s) and a final annealing step (72 °C for 10 min). Details of primer sequences are provided in Supporting Information Table 1. The amplified products were separated on 0.8% agarose gel, and images were acquired and analyzed using Image J software (NIH, Bethesda, MD, USA). Immunofluorescence Staining. Cells treated with multiple doses of Wi-A or 3βmWi-A either alone or followed by various stresses were fixed with chilled absolute methanol and used for antibody immunoblotting as previously described.2 Details of used antibodies and dilutions are provided in Supporting Information Table 2. The images of immunostaining were captured using a Zeiss Axioplan 2 microscope, equipped with a Zeiss AxioCam HRc camera. Immunoblotting. Total protein from control, stressed, and Wi-A/ 3βmWi-A-treated TIG-3 cells was extracted using NP40 lysis buffer followed by centrifugation at 112 RCF for 20 min at 4 °C. Protein lysates (10−20 μg) were used for immunoblotting as previously described.1 Details of antibodies and their dilutions are provided in Supporting Information Table 2. Statistical Analysis. All the experiments were performed in triplicate, and the data were expressed as mean ± SEM. Statistical analyses were performed using Student’s t test or nonparametric Mann−Whitney U-test, whichever was applicable. Statistical significance was defined as p-value ≤ 0.05.

Figure 5. Distinct activities of Wi-A/3βmWi-A and cellular response. Schematic diagram summarizing the differential activities of Wi-A and 3βmWi-A in normal cells and their cellular outcomes.

protected normal cells against Wi-A cytotoxicity to some extent and provided molecular evidence for the long-trusted belief in Ayurveda’s holistic approach of herbal extracts over the purified components. We earlier reported that a leaf extract containing withanone and Wi-A caused selective toxicity to cancer cells.1,2 Here, we provide evidence that an extract containing 3βmWi-A may further protect normal cells against Wi-A toxicity and that 3βmWi-A also possesses antistress properties that may ameliorate the toxicity associated with various targeted therapeutic regimes in clinical practice.

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EXPERIMENTAL SECTION

Wi-A and 3βmWi-A Source. Withanolides were extracted from the alcoholic extracts1 of Withania somnifera leaves by highperformance liquid chromatography (HPLC).1 The compounds (98−99% purity) were used for assays. Cell Culture in Normal and Stressed Conditions. TIG-3 and WI38 (human diploid embryonic lung fibroblasts) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were maintained at 37 °C with 95% O2 and 5% CO2 in a humidified chamber. Presenescent TIG3 cells at PD55 were serially passaged in medium supplemented with subcytotoxic concentrations of either Wi-A (0.1 μM) or 3βmWi-A (0.5 μM) to examine their effect on normal replicative senescence. In order to cause cellular stress, cells were treated with H2O2 (500 μM, 2 h), UV (50 mJ/cm2), doxorubicin (1 μM, 24 h), cisplatin (100 μM, 24 h), and etoposide (100 μM, 24 h) for time periods as indicated. Cell Viability and Proliferation Assay. TIG-3 cells were treated with different concentrations (from 0.15 to 50 μM where indicated) of Wi-A or 3βmWi-A for 48 or 72 h. In stress-protection assays, TIG-3 cells were pretreated with various stresses, as indicated, followed by incubation with either Wi-A or 3βmWi-A. To examine the stressprotection effect of 3βmWi-A, 0.3 μM Wi-A pretreated cells (24 h) were incubated with 1.5 or 3 μM 3βmWi-A for 48 h. In order to examine the comparative cell growth/proliferation, cells were treated with either Wi-A or 3βmWi-A for 48 h followed by H2O2 (500 μM, 2 h) or UV (50 mJ/cm2) treatments followed by recovery (48 h) in respective growth media. The recovered cells were fixed in methanol/ acetone (1:1 v/v), stained with crystal violet vital dye, and visualized under the microscope as described previously.6,22 Cell Morphology Observations. Cell morphology of control and treated cells was captured by a phase contrast microscope (Axioplan 2 Imaging, Carl Zeiss, Inc.) as previously described.6 Colony Formation Assay. Cells (500/well) were seeded in sixwell culture dishes and maintained with regular (every fourth day) change in culture medium (either control, Wi-A, or 3βmWi-A supplemented, as described) until they formed colonies in the next 2 weeks. Colonies were fixed, stained, and counted using a standard

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00573. Supporting Figures S1, S2, and S3; details of used primers, antibodies, and their dilutions in relevant assays in Tables S1 and S2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81 29 861 6713. E-mail: [email protected]. *Tel: +81 29 861 9464. Fax: +81 29 861 2900. E-mail: [email protected]. ORCID

Rajkumar S. Kalra: 0000-0002-8181-457X Sunil C. Kaul: 0000-0002-0046-3916 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by funds from the Department of Biotechnology, Ministry of Science and Technology, New Delhi, India, and the National Institute of Advanced Industrial Science & Technology, Tsukuba, Japan. 2759

DOI: 10.1021/acs.jnatprod.7b00573 J. Nat. Prod. 2017, 80, 2756−2760

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DOI: 10.1021/acs.jnatprod.7b00573 J. Nat. Prod. 2017, 80, 2756−2760