Apoptosis Inducing Activity of Rhinacanthin-C in Doxorubicin-Resistant Breast Cancer MCF-7 Cells

Abstract

Rhinacanthin-C is a natural bioactive naphthoquinone ester with potential chemotherapeutic value in cancer treatment. In this study, we investigated its apoptotic induction ability and the involved mechanisms through the mitogen-activated protein kinases (MAPK) and protein kinase B/glycogen synthase kinase-3β/nuclear factor erythroid 2-related factor 2 (Akt/GSK-3β/Nrf2) signaling pathways in doxorubicin-resistant breast cancer MCF-7 (MCF-7/DOX) cells. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that rhinacanthin-C (3–28 µM) significantly decreased the viability of MCF-7/DOX cells and potentiated hydrogen peroxide cytotoxicity. This naphthoquinone was able to increase intracellular reactive oxygen species (ROS), as measured by the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay. This compound increased the number of apoptotic cells by elevating the ratio of apoptotic checkpoint proteins Bax/Bcl-2 and by decreasing the expression of poly(ADP-ribose) polymerase (PARP) protein. Furthermore, Western blotting analyses showed that treatment with rhinacanthin-C (3–28 µM) for 24 h significantly decreased the expression levels of the phosphorylated forms of MAPK proteins (i.e., extracellular signal regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38), Akt, GSK-3β and Nrf2 proteins in MCF-7/DOX cells. Inhibition of the Akt/GSK-3β/Nrf2 pathway led to a significant reduction in heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADP)(H): quinone oxidoreductase 1 (NQO1) proteins. These findings suggested that rhinacanthin-C was able to induce apoptosis in MCF-7/DOX cells through increased ROS production and suppression of the cell survival systems mediated by the MAPKs and Akt/GSK-3β/Nrf2 signaling pathways.

INTRODUCTION

Chemotherapy is still an essential option in breast cancer treatment. Various cytotoxic drugs, such as doxorubicin and paclitaxel, are often included in the standard chemotherapeutic regimens to kill cancer cells.^1,2) Most cytotoxic drugs are able to produce oxidative damage and apoptotic cell death.³⁾ However, cancer cells may develop drug resistance through several cellular defense mechanisms, which normal cells utilize in their struggle for survival against cellular stress and chemical threats.^4,5) There are many types of adaptive responses involved in the development of drug resistance, such as alteration of apoptotic signals, upregulation of drug efflux transporters and antioxidant enzymes (e.g., reduced nicotinamide adenine dinucleotide phosphate (NADP)(H): quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1)).^6–8) Hence, suppression of cell survival mechanisms can be an effective approach to enhance chemosensitivity and overcome drug resistance in cancer chemotherapy.

The mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) cascades are two major signaling pathways involved in cell survival mechanisms. The activation of MAPKs, including the extracellular signal regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p-38 subfamilies, has been linked to a reduction in stress-induced apoptosis and development of chemoresistance in cancer cells.⁹⁾ Increased expression levels of the anti-apoptotic protein Bcl-2 along with activation of the MAPK pathways have been demonstrated in doxorubicin-resistant K562, MCF-7 and BEL-7402 cancer cells.^10–12) Moreover, activation of the PI3K/Akt cascade was reported to enhance drug resistance in breast cancer cells via a glycogen synthase kinase-3β (GSK-3β)-mediated increase in transcriptional activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of expression of cellular detoxifying and antioxidant enzymes such as NQO1 and HO-1.^7,13) It is very likely that any phytochemicals targeting these signaling pathways can enhance the effectiveness of standard chemotherapeutic regimens in cancer treatment.

Previously, we demonstrated that rhinacanthin-C, a major naphthoquinone ester from Rhinacanthus nasutus (L.) Kurz (Acanthaceae), enhanced doxorubicin cytotoxicity in breast cancer (MCF-7) and doxorubicin-resistant MCF-7 (MCF-7/DOX) cells through direct inhibition of P-glycoprotein (P-gp) function.^14–16) This naphthoquinone exhibited various pharmacological activities, including anti-inflammatory, antifungal, antibacterial, antiviral and cytotoxic activities,^17,18) that contribute to the efficacy of R. nasutus in the treatment of inflammation, hepatitis, and cancer, such as cervical and liver cancers.¹⁹⁾ Rhinacanthin-C has been known to inhibit cell proliferation and induce apoptosis in HeLS3 and KKU-M156 cells, possibly via inhibition of the Akt/nuclear factor-kappaB (NF-κB) or ERK1/2 signaling pathway.^20,21) However, the ability of rhinacanthin-C to overcome drug resistance in breast cancer cells through promotion of oxidative stress and suppression of the cellular detoxification system has not been clarified.

This study investigated the apoptosis-inducing effect of rhinacanthin-C in doxorubicin-resistant breast cancer cells (MCF-7/DOX) and its underlying mechanisms. Our results demonstrated that MAPK and Akt/GSK-3β signaling in resistant cancer cells could be important targets for rhinacanthin-C-mediated apoptosis. Rhinacanthin-C-mediated inhibition of MAPK signaling might enhance oxidative damage and promote apoptotic cell death. Furthermore, its inhibitory effect on Akt/GSK-3β signaling in these resistant cells decreased the Nrf2-regulated expression of antioxidant and detoxifying enzymes, the primary defense mechanism against oxidative stress. These findings obviously support the potential value of rhinacanthin-C in chemotherapeutic regimens for cancer treatment.

MATERIALS AND METHODS

Chemicals and Antibodies

Rhinacanthin-C (RN-C) (purity >94%) (Fig. 1) was isolated and purified from the roots of R. nasutus, as previously described.¹⁹⁾ Doxorubicin hydrochloride (DOX) was purchased from Abcam (Cambridge, U.K.). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and Hoechst 33342 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco Life Technologies (Grand Island, NY, U.S.A.). Hydrogen peroxide (H₂O₂), fetal bovine serum (FBS) and Immobilon-P polyvinylidene fluoride (PVDF) membranes were purchased from Merck Millipore (Darmstadt, Germany). Mouse monoclonal anti-Nrf2 (sc-365949), anti-NQO1 (sc-32793), anti-HO-1 (sc-136960), anti-Bcl-2 (sc-7382), anti-Bax (sc-7480), anti-P-gp (sc-55510), and anti-poly(ADP-ribose) polymerase (PARP) (sc-8007) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Mouse monoclonal anti-p-ERK1/2 (#5726), anti-ERK1/2 (#4696), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#AP308P) antibodies were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Secondary goat anti-mouse immunoglobulin G (IgG) (H&L) conjugated to horseradish peroxidase (HRP) (#AP124P) was purchased from Calbiochem (San Diego, CA, U.S.A.). Rabbit monoclonal anti-p-Akt (#9271), anti-Akt (#9272), anti-p-JNK (#4668), anti-JNK (#9252), anti-p-GSK-3β (#9323), anti-GSK-3β (#9315), anti-p-p38 (#9215) and anti-p38 (#8690) antibodies and rabbit monoclonal HRP-conjugated anti-rabbit-IgG antibodies were purchased from Cell Signaling Technology (Beverly). The rabbit monoclonal anti-p-Nrf2 (P00078) was purchased from Boster Bio (Pleasanton, CA, U.S.A.). Super Signal^® West Pico Chemiluminescent Substrate was purchased from Pierce Biotechnology (Rockford, IL, U.S.A.).

Fig. 1. Chemical Structure of Rhinacanthin-C

Cell Cultures

The human breast adenocarcinoma MCF-7 (ATC C^@ HTB-22™) cell line was obtained from the American Type Culture Collection (ATC C, Rockville, MD, U.S.A.) and further developed into the doxorubicin-resistant subline MCF-7/DOX cells by overexpressing P-gp (Fig. 2A), as previously described.^14,22,23) MCF-7/DOX cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin and 1.5 µM doxorubicin at 37 °C in a humidified atmosphere of 5% CO₂. One week before the experiments, MCF-7/DOX cells were cultured in DOX-free RPMI-1640 complete medium.

Fig. 2. Protein Expression and Activity of P-gp in MCF-7 and MCF-7/DOX Cells

(A) Immunoblots and densitometric analysis of P-gp and GAPDH (an internal control). (B) Cell viability after 48-h of exposure to rhinacanthin-C.

Cell Viability Assay

Cell viability was evaluated using the MTT colorimetric assay. Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and incubated at 37 °C overnight. Then, the cells were treated with rhinacanthin-C at different concentrations (0–30 µM) for 48 h. Following the treatment, the cells were incubated with serum-free medium containing MTT reagent (0.5 mg/mL) for 4 h at 37 °C. The formazan crystals produced by viable cells were solubilized with dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader (Wallac 1420 VICTOR 3, PerkinElmer, Inc., Hopkinton, MA, U.S.A.). The effect of rhinacanthin-C on oxidative stress-induced cell death was also investigated in MCF-7/DOX cells. The cells were incubated with H₂O₂ (50 µM) for 1 h prior to the addition of rhinacanthin-C (3, 16, and 28 µM). At the end of the 24-h treatment period, cell viability was determined by an MTT assay, as described above.

Apoptosis Assay

Apoptotic cells were assessed using the fluorescent nuclear staining dye Hoechst 33342.²⁴⁾ MCF-7/DOX cells (8 × 10³ cells/well) were seeded in 96-well plates overnight. Then, the cells were treated with rhinacanthin-C for 24–48 h. Following the treatment, the cells were incubated with Hoechst 33342 (10 µg/mL) for 30 min in the dark. Morphological changes, i.e., nuclear chromatin condensation and DNA fragmentation of apoptotic cells, were visualized under a fluorescence microscope (20×, original magnification) (BX-FLA, Olympus, Tokyo, Japan) at excitation/emission wavelengths of 350/461 nm. Apoptotic cells were counted and are presented as a percentage of the total cells.

Determination of Reactive Oxygen Species (ROS)

Cellular ROS was measured by the DCFH-DA assay.²⁵⁾ MCF-7/DOX cells (2 × 10⁴ cells/well) were grown in 96-well plates overnight. Then, the cells were incubated with 100 µM DCFH-DA for 30 min prior to the addition of either rhinacanthin-C or hydrogen peroxide (H₂O₂; 250 µM) as a positive control for 1 h. At the end of the treatment period, the cells were lysed with 1% Triton X-100 and the fluorescence intensity of DCF was measured at 485/535 nm (excitation/emission) using a microplate reader.

Western Blotting Analysis

The levels of protein markers related to apoptosis and signaling proteins in the MAPK and Akt/GSK-3β/Nrf2 pathways were determined by Western blot analysis. Following the treatment, the cells were harvested and total cellular protein was extracted as described previously.¹⁴⁾ Equal amounts of protein sample (30 µg) were separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to a PVDF membrane. After blocking with 5% skim milk, the membranes were incubated with primary antibodies (1 : 1000) at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody (1 : 2000) for 1 h at room temperature and developed with the Super Signal^® West Pico chemiluminescent substrates kit according to the manufacturer’s instructions. The signals were captured using a luminescence-image analyzer (Image Quant™ LAS 4000, GE Healthcare Biosciences, Japan). The density of protein bands was quantified using ImageJ software (NIH, Bethesda, MD, U.S.A.) and normalized to that of GAPDH (an internal control).

Statistical Analysis

Data are expressed as the mean ± standard error of the mean (S.E.M.) of 3–4 independent experiments. One-way ANOVA, followed by Tukey’s post hoc analysis, was performed for multiple comparisons, whereas Student’s t-test was used for two-group comparisons. p < 0.05 was considered significant.

RESULTS

Cytotoxicity of Rhinacanthin-C in Doxorubicin-Resistant (MCF-7/DOX) Cells

As shown by the IC₅₀ values obtained from the MTT assay in Table 1, the MCF-7/DOX cells were approximately 62-fold more resistant to DOX than their parental MCF-7 cells. Rhinacanthin-C was able to reduce the viability of both MCF-7 and MCF-7/DOX cells in a concentration-dependent manner (Fig. 2B). Moreover, both cell lines exhibited comparable sensitivity to rhinacanthin-C after 48-h of treatment, with IC₅₀ values of 11.22 ± 1.41 µM (for MCF-7 cells) and 12.63 ± 1.31 µM (for MCF-7/DOX cells) (Table 1).

Table 1. The IC₅₀ Values of Test Compounds in MCF-7 and MCF-7/DOX Cells after 48-h Treatment

Compound	IC50 in MCF-7 cells (µM)	IC50 in MCF-7/DOX cells (µM)	Resistance index (fold)
Doxorubicin	2.52 ± 1.55	155.85 ± 1.04	62
Rhinacanthin-C	12.63 ± 1.31	11.22 ± 1.41	0.9

Resistance index was calculated from the ratio between the IC₅₀ value of each test compound obtained from MCF-7/DOX cells to that from MCF-7 cells. Data are expressed as the means ± standard error of the mean (S.E.M.) (n = 4).

At 24-h of treatment, neither rhinacanthin-C (at 3, 16, and 28 µM) nor H₂O₂ (at 50 µM) was toxic to MCF-7/DOX cells, as evidenced by their >80% cell viability (Fig. 3A). The combination of rhinacanthin-C and H₂O₂ markedly increased cytotoxicity in MCF-7/DOX cells compared with each compound alone (Fig. 3A). Increasing the concentration of rhinacanthin-C from 3 µM to 28 µM in the combinatorial regimens resulted in a reduction in cell viability from 62.85 to 38.55%.

Fig. 3. (A) Effect of Rhinacanthin-C on MCF-7/DOX Viability in the Presence of Hydrogen Peroxide (H₂O_2; 50 µM) after 24-h of Exposure; (B) ROS Production in MCF-7/DOX Cells after 1-h of Treatment with Rhinacanthin-C or Hydrogen Peroxide (H₂O₂; 250 µM)

* p < 0.05 compared with the untreated group (black solid bar; ). ^#p < 0.05 compared with the H₂O₂ alone group (dark gray solid bar; ).

Effect of Rhinacanthin-C on Intracellular ROS in MCF-7/DOX Cells

An increase in ROS generation was observed in MCF-7/DOX cells after 1-h of exposure to rhinacanthin-C, as measured by the DCFH-DA assay (Fig. 3B). At a concentration of 3 µM, rhinacanthin-C significantly increased cellular ROS by 3.1-fold compared to the untreated control group. Upon increasing the concentration of rhinacanthin-C to 16 and 28 µM, the levels of ROS were reduced to 1.7 fold higher than those of the untreated group (Fig. 3B). Nevertheless, these findings suggested that rhinacanthin-C was able to induce oxidative stress in doxorubicin-resistant breast cancer cells through ROS production.

Effects of Rhinacanthin-C on Apoptosis Induction and Expression of Bcl-2 and Poly(ADP-ribose) Polymerase (PARP) Proteins

As shown in Fig. 4A, a significant number of MCF-7/DOX cells underwent apoptosis after exposure to rhinacanthin-C for 24 and 48 h, as measured by the Hoechst 33342 staining assay. The induction of apoptosis by rhinacanthin-C was concentration- and time-dependent. After 24-h of exposure to rhinacanthin-C, the percentages of apoptotic MCF-7/DOX cells increased from 15.3 to 43.6% when the concentration was increased from 3 to 28 µM (Fig. 4B). Moreover, when the treatment period was extended to 48 h, the percentages of apoptotic cells increased by approximately 1.7- to 2.2-fold.

Fig. 4. Rhinacanthin-C Induced Apoptosis and Alteration of Apoptosis-Related Proteins in MCF-7/DOX Cells after 24–48 h of Exposure

(A) Images of apoptotic cells (i.e., cells having condensed chromatin and/or fragmented nuclei) visualized under a fluorescence microscope (20× magnification; scale bar = 200 µm). (B) The percentages of apoptotic cells relative to total cells. (C) Immunoblots and densitometric analysis of Bax and Bcl-2 proteins and (D) PARP protein in MCF-7/DOX cells after treatment with rhinacanthin-C for 24 h. * p < 0.05 compared with the untreated group.

The expression levels of the pro-apoptotic Bax and the anti-apoptotic Bcl-2 proteins, along with the PARP protein, in MCF-7/DOX cells were determined after 24-h of treatment with rhinacanthin-C at 3 different concentrations. Our Western blot analyses showed that rhinacanthin-C significantly increased the Bax/Bcl-2 ratio and suppressed PARP expression in a concentration-dependent manner (Figs. 4C, D). The Bax/Bcl-2 ratio in the cells treated with rhinacanthin-C at 28 µM was approximately 2.4-fold higher than that in the untreated control group. In addition, the extent of PARP was reduced by 70% from that of the untreated control.

Effects of Rhinacanthin-C on the MAPKs Signaling Pathway

We further investigated the effects of rhinacanthin-C on the expression levels of phosphorylated signaling proteins in the MAPK pathway, including ERK1/2, JNK and p-38. The MAPK signaling pathway has been associated with the cell stress response, cell survival and apoptosis mechanisms.⁹⁾ Upon treatment with rhinacanthin-C for 24 h, the expression levels of the phosphorylated ERK1/2, JNK and p-38 proteins in the MCF-7/DOX cells were significantly lower than those in the untreated control group (Figs. 5A–C). Thus, low concentrations (3–16 µM) of rhinacanthin-C were able to suppress the activities of MAPKs in doxorubicin-resistant MCF-7 cells after a 24-h treatment period.

Fig. 5. Immunoblots and Densitometric Analysis

(A) ERK1/2, (B) JNK, (C) p-38 and their phosphorylated forms in MCF-7/DOX cells after treatment with rhinacanthin-C for 24 h. * p < 0.05 compared with the untreated group.

Effects of Rhinacanthin-C on the Akt/GSK-3β/Nrf2 Pathway

In this study, we also determined the effects of rhinacanthin-C on cellular detoxification processes through the Akt/GSK-3β/Nrf2 pathway. It has been reported that the Akt/GSK-3β/Nrf2 cascade is involved in chemoresistance through the induction of several antioxidant and detoxifying enzymes, such as NQO1 and HO-1, in response to stresses.^24,26) As shown in Figs. 6A and B, even at a low concentration of 3 µM, rhinacanthin-C could significantly reduce Akt and GSK-3β phosphorylation in MCF-7/DOX cells after a 24-h treatment period. The levels of phosphorylated forms of both proteins were markedly decreased when the concentration of rhinacanthin-C was increased to 16 µM. Moreover, our results demonstrated that phosphorylated Nrf2 protein, the well-known downstream effector of the Akt/GSK-3β signaling cascade, was expressed at a lower degree in rhinacanthin-C-treated cells than in the untreated control group.

Fig. 6. Immunoblots and Densitometric Analysis

(A) Akt, GSK-3β and their phosphorylated forms, (B) Nrf2 and phosphorylated Nrf2 in MCF-7/DOX cells after treatment with rhinacanthin-C for 24 h. * p < 0.05 compared with the untreated group.

Effects of Rhinacanthin-C on the Expression Levels of NQO1 and HO-1

The activity of the transcription factor Nrf2 is essential in regulating the expression of antioxidant and detoxifying enzymes such as NQO1 and HO-1.^13,27,28) Figure 7 shows that, after 24-h of exposure, rhinacanthin-C at 16 and 28 µM significantly reduced HO-1 and NQO1 expression in the MCF-7/DOX cells by approximately 37 and 54%, respectively, compared with the untreated control group.

Fig. 7. Expression of NQO1 and HO-1 in MCF-7/DOX Cells after Treatment with Rhinacanthin-C for 24 h

* p < 0.05 compared with the untreated group.

DISCUSSION

Resistance to chemotherapy has been one of the major problems in cancer treatment. Cancer cells can develop multidrug resistance (MDR) and survive against cellular stresses or chemical threats through various mechanisms, such as an increased number of drug efflux transporters, altered apoptotic pathways and disturbed cellular detoxification processes.^3,29) In this study, we demonstrated for the first time that rhinacanthin-C, a major naphthoquinone derivative from R. nasutus, was able to induce cytotoxicity against doxorubicin-resistant MCF-7 breast cancer cells with a high expression level of P-gp to a comparable degree as doxorubicin-sensitive MCF-7 cells. These results supported our previous findings that despite its intrinsic ability to inhibit P-gp function, rhinacanthin-C was not a substrate of P-gp.^14–16) This naphthoquinone was approximately 14-fold more potent than doxorubicin in reducing the viability of resistant MCF-7/DOX cells after 48-h of treatment. In addition, rhinacanthin-C was able to produce intracellular ROS and potentiate H₂O₂ toxicity. Although the cytotoxic activity of rhinacanthin-C was concentration-dependent, the elevated ROS levels as measured by the DCFH-DA assay were not well correlated with the increasing rhinacanthin-C concentration. Regarding this, it is possible that DCFH-DA, which is more specific toward H₂O₂,^30,31) could not effectively detect other types of ROS produced with the increasing rhinacanthin-C concentration. The contribution of rhinacanthin-C-generated ROS in killing MCF-7/DOX cells should be further investigated. Moreover, the direct effect of this naphthoquoinone on the apoptosis induction pathway could not be excluded. Obviously, rhinacanthin-C was able to induce apoptotic cell death with its intrinsic ability to suppress the detoxification processes of doxorubicin-resistant cells.

Various approaches to overcoming MDR in cancer therapy have been introduced to effectively improve chemotherapeutic outcomes. In addition to suppression of P-gp function, the induction of apoptosis, particularly in resistant cancerous cells, could be an effective MDR reversal strategy.^29,32) Previously, we demonstrated that rhinacanthin-C could inhibit P-gp activity in MCF-7 and MCF-7/DOX cells, leading to increased cell chemosensitivity to doxorubicin.¹⁴⁾ In this study, we further demonstrated the effectiveness of rhinacanthin-C in inducing apoptosis of resistant MCF-7/DOX cells within 24–48 h after exposure. The increase in apoptotic cell death could result from an increase in the ratio of the apoptotic checkpoint proteins Bax/Bcl-2 as well as a decrease in the expression of PARP, a DNA repair protein. Anti-apoptotic Bcl-2 family proteins are highly expressed in cancer cells with an MDR phenotype as an adaptive response to sustain their viability and give rise to chemotherapy resistance.^12,33) An increase in the Bax/Bcl-2 ratio subsequently results in activation of caspase activity, an increase in DNA damage and apoptotic cell death.^10,34,35) Rhinacanthin-C has been reported to cause G2/M cell-cycle arrest and caspase-3 activation in HeLaS3 cells, resulting in apoptosis.¹⁸⁾

Suppression of the detoxifying processes related to the MAPK and Akt/GSK-3β/Nrf2 signaling pathways might contribute to rhinacanthin-C-mediated apoptosis. In response to cell stress signals, activation of MAPKs, including ERK1/2, JNK and p-38, is essential for cell growth, proliferation, and differentiation, leading to a decrease in apoptotic cell death.^9,36–40) However, hyperactivation of the MAPK pathway has been associated with the development of MDR in cancer cells.^9,39,41,42) Our results showed that rhinacanthin-C was able to inhibit the sustained activation of the MAPK signaling pathway, as evidenced by the reduction in phosphorylated forms of ERK1/2, JNK and p-38 in MCF-7/DOX cells after 24-h of exposure, followed by a significant increase in the number of apoptotic cells. Concurrently, rhinacanthin-C significantly reduced the amount of phosphorylated Akt, GSK-3β and Nrf2 in rhinacanthin-C-treated MCF-7/DOX cells. Moreover, the expression levels of HO-1 and NQO1 in these cells were significantly decreased. These findings suggested that, in addition to the MAPK pathway, rhinacanthin-C was able to inhibit the sustained activation of the Akt/GSK-3β/Nrf2 signaling pathway in doxorubicin-resistant MCF-7 cells. Nrf2 is an essential transcription factor that fights oxidative stress at the cellular level by regulating the expression of antioxidant and detoxifying enzymes such as HO-1 and NQO1.^13,27,28) The phosphorylation of the repressor GSK-3β at serine (Ser) 9 by Akt reduces Nrf2 phosphorylation so that Nrf2 can escape the ubiquitination and proteasomal degradation processes in the cytoplasm.^13,26) Hence, Nrf2 translocates into the nucleus, and its transcriptional activity increases, making the cells resistant to stress and apoptotic signals.^13,43) Similar to the MAPK pathway, high Akt/GSK-3β/Nrf2 basal activities with overexpression of HO-1 and NQO1 were detected in MDR cancer cells such as MCF-7/DOX, BEL-7402/ADM and A549/DDP cells.^11,44,45) It has been demonstrated that some phytochemicals such as baicalin, chrysin and luteolin-7-O-glucoside were able to promote apoptosis in resistant cancer cells via inhibition of the Akt/GSK-3β/Nrf2 signaling cascade.^11,46,47) Our results indicated that rhinacanthin-C could suppress the expression of the antioxidant and detoxifying enzymes HO-1 and NQO1 via inhibition of the Akt/GSK-3β/Nrf2 pathway. The collective inhibitory effect of rhinacanthin-C on cellular defense mechanisms through the MAPKs and Akt/GSK-3β/Nrf2 signaling cascades contributed in part to promoting stress-mediated apoptosis in doxorubicin-resistant MCF-7 cells. The potential effects of this naphthoquinone on other redox-sensitive transcription factors, such as NF-κB, activator protein-1 (AP-1) and hypoxia-inducible factor 1 (HIF-1), as well as other survival pathways, such as the expression of efflux transporters, should be investigated further.

In conclusion, rhinacanthin-C was able to induce apoptosis in doxorubicin-resistant breast cancer MCF-7 cells through ROS production and suppression of the cell survival systems mediated by the MAPK and Akt/GSK-3β/Nrf2 signaling pathways. Inhibition of MAPK signaling affected the ratio of the checkpoint proteins Bax/Bcl-2 in favor of apoptosis induction. In addition, rhinacanthin-C might increase cellular stress and promote apoptosis via inhibition of the Akt/GSK-3β/Nrf2 pathway, leading to downregulation of the antioxidant and detoxifying enzymes NQO1 and HO-1. These findings suggested that rhinacanthin-C may have potential value in chemotherapeutic regimens even against drug-resistant cancer cells.

Acknowledgments

This work was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). A Scholarship to Suwichak Chaisit from the Graduate School, Chulalongkorn University to commemorate The Celebration on the Auspicious Occasion of Her Royal Highness Princess Maha Chakri Sirindhorn’s 5th Cycle (60th) Birthday is gratefully acknowledged. We also thank Dr. Pongpun Siripong, National Cancer Institute, Bangkok, Thailand for kindly providing rhinacanthin-C in this study.

Conflict of Interest

The authors declare no conflict of interest.

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