Chemotherapy is an effective way to manage many types of cancer; however, the prospects for a complete cure are limited by the emergence of primary and acquired drug resistance to chemotherapeutic agents [1
]. Cancer cells develop multidrug resistance (MDR) through multiple mechanisms, including decreased drug uptake, increased drug efflux, activation of the DNA damage response, and enhanced cell survival [1
]. For example, overexpression of ATP-binding cassette (ABC) transporter family proteins, such as P-glycoprotein (MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1), enhances efflux of anti-cancer drugs from cells [1
]. Cancer cells can also acquire drug resistance by stimulating the repair of drug-induced DNA damage and inhibiting cell death signaling [3
Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent enzyme that deacetylates various histone and non-histone substrates, such as p53, c-MYC, and FOXO, to regulate key biological processes, including DNA repair, cell cycle, apoptosis, cell survival, cellular metabolism, and cell senescence [4
]. SIRT1 expression is upregulated in some cancers, such as prostate and colon cancer, and downregulated in others, such as breast cancer and hepatic cell carcinoma, suggesting that SIRT1 has different roles depending on cellular context [6
]. Nonetheless, growing evidence implicates SIRT1 in cancer promotion and development of resistance to chemotherapeutical agents, including cisplatin, doxorubicin, and camptothecin [7
]. Inhibition of SIRT1 decreases growth and viability of cancer cells while its overexpression impairs apoptosis, suggesting that SIRT1 is a critical regulator of cell proliferation and survival [7
]. In addition, siRNA-mediated depletion of SIRT1 re-sensitizes cisplatin-resistant cancer cells to cisplatin [10
]. Therefore, modulation of SIRT1 activity could be a viable strategy for overcoming MDR.
Ginsenosides, the active components of Panax ginseng C.A. Meyer
, display anti-cancer properties [11
] and reverse drug resistance. In mice implanted with adriamycin-resistant leukemia cells, ginsenosides Rg3 inhibit MDR1 activity by reducing membrane fluidity [13
]. In adriamycin-resistant breast cancer cells, Rh2 attenuates adriamycin resistance by inhibiting MDR1 activity and increasing the rate and amount of adriamycin entering cellular/subcellular compartments [14
]. We have previously reported that Rp1, a novel ginsenoside derivative, reverses MDR by downregulating MDR1 expression and inhibiting Src [2
]. Aside from the effects on MDR1, the molecular mechanisms underlying ginsenoside modulation of chemosensitivity remain unclear. Actinomycin D (ActD) is an anti-cancer drug, which blocks DNA-dependent transcription and inhibits topoisomerase, resulting in DNA double stand breaks. Although ActD, an FDA-approved chemotherapeutic drug, has been used for treatment of various cancers, its use is limited by its toxicity at high dose [15
]. For a beneficial use of ActD, combination therapy using a low dose should be exploited. Accordingly, in the present study, we intended to assess the anti-cancer effects of ginsenoside Rp1, ActD, and their co-administration in drug-resistant cells and murine xenograft model of colon cancer, and investigate the underlying mechanisms.
Although conventional chemotherapy is widely used for the treatment of cancer, anti-cancer agents are often toxic not only to tumor cells but also to healthy cells [29
]. Worse still, cancer cells can transition from a chemotherapy-sensitive to a chemotherapy-resistant phenotype, which is a major cause of treatment failure and metastasis [3
]. Thus, it is very important to explore novel strategies to overcome MDR. Several mechanisms are involved in drug resistance, including enhanced efflux of chemotherapeutic agents from cancer cells, activation of cell survival signaling, and inhibition of apoptosis. This study provides in vitro and in vivo evidence for synergistic inhibitory action of ginsenoside Rp1 with ActD on the growth of drug-resistant cancer cells. We demonstrated that these effects are exerted through inactivation of the AKT/SIRT1 pathway and increase in p53 acetylation.
Natural compounds with anticancer properties have generated considerable interest among researchers owing to their safety and efficacy [29
]. Ginsenosides, the main active compounds extracted from P. ginseng
, display a wide range of therapeutic and pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer effects [30
]. Rg3, one of the active ginsenosides, exerts antitumor effects in various cancers through induction of apoptosis and inhibition of proliferation, metastasis, angiogenesis, and MDR [31
]. We previously reported that Rp1 inhibits MDR through downregulation of MDR1 and modulation of lipid rafts [2
]. In this study, we further elucidated the mechanisms of Rp1 modulation of drug sensitivity, focusing on the AKT-SIRT1-p53 pathway.
Recently, an association between SIRT1 expression and drug resistance has been reported [4
]. SIRT1 regulates various biological functions, such as cell proliferation, cell survival, immune response, and carcinogenesis. Although the roles of SIRT1 in cancer are controversial, its overexpression is a feature of many solid tumors and hematopoietic malignances [32
]. Our analysis of publicly available Kaplan–Meier data revealed that SIRT1 expression is negatively correlated with survival in several human cancer types (Figure 7
). Moreover, accumulating evidence suggests that SIRT1 is activated in multidrug-resistant cell lines. SIRT1 positively regulates the expression of ABC transporters, such as MDR1 and ABCA1, which promote efflux of anti-cancer drugs from cells [35
]. We observed that, upon ActD treatment, SIRT1 was upregulated in drug-resistant LS513, OVCAR-DXR, and A549-DXR cells, and downregulated in drug-sensitive SW620 cells (Figure 2
, Figure 3
, and Figure S1
). SIRT1 upregulation is not limited to ActD-treated cells, as paclitaxel caused a similar effect in LS513 cells (Figure 2
B). In addition, SIRT1 inhibition, either pharmacological or through gene knockdown, augmented ActD-induced cell growth inhibition and PARP cleavage, suggesting that SIRT1 upregulation is involved in resistance to ActD (Figure 3
). Therefore, it is interesting that Rp1 in combination with ActD, but not Rp1 alone, downregulated SIRT1 (Figure 2
A). When SIRT1 was overexpressed, Rp1 was unable to re-sensitize cells to ActD, suggesting that upregulation of SIRT1 plays an important role in drug resistance (Figure 2
D). SIRT1-mediated drug resistance appeared not to be related to MDR1 expression because SIRT1 knockdown had a limited effect on MDR1 expression in LS513 cells.
Certain chemotherapeutic agents, including ActD, exert their cytotoxic effects by damaging the DNA. Cancer cells are able to boost alternative DNA repair pathways to avoid apoptosis, thus developing drug resistance [36
]. SIRT1 deacetylates several master transcriptional factors involved in apoptosis and DNA damage, including p53, to inhibit apoptosis [21
]. SIRT1-deficient cells exhibit p53 hyperacetylation following DNA damage [37
]. In LS513 cells, although ActD could upregulate p53 expression, p53 acetylation was minimal, probably due to ActD-induced SIRT1 upregulation (Figure 3
B,C). When SIRT1 was inhibited, either by EX527 or siRNA-mediated silencing, p53 acetylation and PARP cleavage increased upon ActD treatment (Figure 3
B,C). Rp1 attenuated ActD-induced SIRT1 upregulation to increase p53 acetylation, leading to synergistic anti-cancer effects in drug-resistant cells (Figure 6
Bhatia et al. reported that pharmacological inhibition of SIRT1 or SIRT1 knockdown increase apoptosis in leukemia stem cells, and that the inhibitory effects of SIRT1 depend on p53 expression and acetylation [38
]. SIRT1 can prevent oxidative stress-induced apoptosis in mesangial cells through p53 deacetylation [39
]. Thus, we investigated whether the SIRT1-p53 deacetylation pathway is important for ActD-mediated cell death. ActD treatment enhanced PARP cleavage and p53 acetylation in SIRT1-deficient LS513 cells (Figure 4
A). However, ActD could not increase PARP cleavage when both SIRT1 and p53 were knocked down even though ActD-induced DNA damage (assessed by γ-H2AX levels) was similar to that in SIRT1-depleted cells (Figure 4
A). In line with these results, SIRT1 knockdown re-sensitized p53-expressing HCT-116 cells, but not p53-deficient HCT-116 cells, to ActD (Figure 4
B,C), implying that p53 activation through SIRT1 inhibition is critical for ActD-induced cell death.
The PI3K/AKT pathway is often activated in cancer and contributes to tumorigenesis, metastasis, and chemoresistance [40
]. Recently, PI3K/AKT signaling has been reported to modulate chemoresistance by regulating ABGG2 expression in human multiple myeloma [42
]. In our study, ActD promoted SIRT1 upregulation as well as AKT phosphorylation (Figure 5
C). It is possible that SIRT1 upregulation contributes to ActD-induced AKT activation because SIRT1 is known to deacetylate AKT. Deacetylation of AKT is necessary for its binding to PIP3 and, in turn, its membrane localization and activation [25
]. However, SIRT1 knockdown decreased AKT phosphorylation only to a small extent. AKT inactivation, either through siRNA or using LY294002, a PI3K/AKT inhibitor, deceased SIRT1 levels and re-sensitized LS513 cells to ActD (Figure 5
B–E), implying that SRIT1 could be a downstream target of AKT.
An intact structure of lipid rafts, cholesterol-enriched membrane microdomains, is critical for AKT activation and, thus, cell survival [28
]. Exposure to ActD stimulated AKT activity and SIRT1 levels, which was attenuated by addition of Rp1 (Figure 6
B). Rp1 (5 μM) alone had a minimal effect on cell viability, but when combined with 30 nM ActD, the two agents acted synergistically to induce cell death through AKT inactivation and downregulation of SIRT1 (Figure 6
B). It is possible that co-treatment with Rp1 and ActD amplifies modification of lipid rafts, leading to AKT inactivation and drug sensitivity. In support of this notion, treatment with a lipid-raft-disrupting agent, MβCD, caused AKT inactivation and SIRT1 downregulation in LS513 cells (Figure 6
C). Cholesterol addition, which is known to fortify lipid rafts and increase AKT activation [28
], could preserve SIRT1 levels and thus reverse drug sensitivity induced by Rp1 and ActD co-treatment (Figure 6
), implicating lipid rafts in Rp1-medated drug sensitivity.
4. Materials and Methods
Ginsenoside Rp1 (purity, 99%), a gift from Ambo Institute (Seoul, Korea), was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) [43
]. ActD was purchased from Sigma-Aldrich. Anti-MDR1, anti-SIRT1, normal rabbit and mouse IgG, HRP-conjugated rabbit, and mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-H2AX (Ser139, γ-H2AX) antibody was obtained from Millipore Corporation (Bedford, MA, USA). Antibodies against PARP, acetyl p53, phospho-SIRT1, phospho-AKT, and AKT were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody was obtained from Sigma-Aldrich.
4.2. Cell Culture
Human colorectal cancer cell lines, LS513 (drug resistant) and SW620 (lymph node metastasis) were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and the American Type Culture Collection (ATCC, Rockville, MD, USA), respectively. Human colon cancer cells, HCT116 p53 WT (wild type p53) and HCT116 p53-null (p53 null mutant) are a kind gift from Dr. Sung-Ho Goh (National Cancer Center, Goyang, Korea).
HCT116 p53WT, HCT116 p53-null, SW620, and LS513 cells were grown in RPMI 1640 medium with L-glutamine (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, and 100 µg/mL streptomycin (Antibiotics-Antimycotic, Gibco Laboratories Co., Grand Island, NY, USA) as previously described [2
]. OVCAR-DXR cells were grown under selective pressure of 1 µM doxorubicin. The cells were allowed to adhere overnight and reach approximately 70% confluence. Before treatment, the cells were serum starved for 4 h using RPMI 1640 medium with 0.1% bovine serum albumin (BSA; USB Corp., Cleveland, OH, USA).
4.3. Cell Viability and Proliferation Assays
For the cell viability assay, cells were exposed to indicated concentrations of reagents in RPMI 1640 medium containing 0.1% BSA. Following incubation, the cells were stained with green-fluorescent calcein AM and red-fluorescent ethidium homodimer-1. For the cell proliferation assay, LS513 cells (5 × 104
cells/well) were plated in a 96-well culture plate for 24 h before treatment (approximately 70% confluence). Cell growth was determined using the CellTiter 96 Kit (MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega, Madison, WI, USA) as previously described [2
4.4. siRNA Transfection
SIRT1 plasmid was kindly provided by Dr. Dong Hoon Shin (National Cancer Center, Korea). Reverse transfection of siRNA duplexes into cells was performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) while transfection of plasmids into cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The sequences of siRNA for the negative control (NC), SIRT1, and AKT were 5′-CCUACGCCACCAAUUUCGU-3′ (NC, Bioneer, Daejeon, Korea) and 5′-CUAAUCUAGACCAAAGAAU-3′ (SIRT1, Bioneer) 5′-GACAACCGCCAUCCAGACU (AKT, Bioneer), respectively.
4.5. Immunoblotting Analysis
Cells were lysed with 2 x SDS lysis buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 1 mM Na3
, 1 mM DTT, 2% SDS, 20% glycerol) and boiled for 5 min, followed by protein assay to determine protein concentration of each sample using Micro BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Total cellular protein (20 μg) was separated by 8 or 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked at room temperature (RT) in tris-buffered saline and tween 20 (TBS-T) containing 5% non-fat dried milk. The membranes were incubated with the primary antibody overnight at 4 ˚C, washed two times with TBS-T for 30 min, incubated with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies for 1 h at RT, and then washed with TBS-T two times for 15 min. The labeled proteins were visualized by the enhanced chemi-luminescence method. The levels of protein were quantified by a densitometry and normalized to loading control β-actin or GAPDH. Densitometry readings maybe found in Supplemental data
4.6. Immunofluorescence and Confocal Microscopy Imaging
Cells were grown on to glass cover slips and fixed with 2% paraformaldehyde in PBS. The fixed cells were rinsed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 3% BSA in PBS for 1 h at room temperature. Subsequently, the cells were incubated with indicated primary antibodies or non-specific IgG overnight at 4 ˚C, washed in PBS, and then exposed to Alexa 568-conjugated secondary IgG for 1 h at room temperature, respectively. The cells were stained with Hoechst 33342 before the glass cover slips were washed in PBS and mounted on glass slides. The cells were examined under the Zeiss LSM 510-Meta confocal fluorescence microscope (Carl Zeiss, Jena, Germany).
4.7. Flow Cytometric Analysis of Cell Death
Cells were harvested, fixed with 70% ethanol, and stained with propidium iodide (PI) solution (20 µg/mL PI, 0.1% sodium citrate, 50 µg/mL RNase A, 0.03% NP-40, PBS) before they were analyzed by flow cytometry with CellQuest software (BD Biosciences, San Jose, CA, USA).
4.8. Establishment of a Murine Xenograft Model
LS513 cells (3 × 106) were suspended in PBS, mixed with Matrigel (1:1, v/v; BD Biosciences, Bedford, MA, USA), and then injected subcutaneously into 6-week-old Balb/c nude mice (0.1 mL per animal). Tumor length (L) and width (W) were measured using a caliper, and the average tumor volume was calculated as (L × W2)/2. When the tumors reached an average volume of ~100 mm3, the mice were randomized into four groups, with 3 mice per group. The mice were treated by intraperitoneal injection twice a week either with PBS (as a control), ActD, Rp1, or both ActD and Rp1. The mice in both control group (PBS-treated) and drug-treated group were fed with a standard chow diet (altromin 1314; Altromin, Lage, Germany). Tumor size was measured with calipers. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Cancer Center Research Institute (NCCRI). NCCRI is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility and abides by the Institute of Laboratory Animal Resources (ILAR) guide.