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. 2022 Nov 18;11(2):83–90. doi: 10.1016/j.prnil.2022.11.003

A novel biguanide derivative, IM176, induces prostate cancer cell death by modulating the AMPK-mTOR and androgen receptor signaling pathways

Yunlim Kim a,1, Sangjun Yoo b,1, Bumjin Lim c, Jun Hyuk Hong c, Cheol Kwak d, Dalsan You c,∗∗∗, Jung Jin Hwang a,∗∗, Choung-Soo Kim e,∗
PMCID: PMC10318334  PMID: 37409095

Abstract

Background

Metformin and phenformin, biguanide derivatives that are widely used to treat type 2 diabetes mellitus, have recently been shown to exert potential anticancer effects in prostate cancer. This study compared the antiprostate cancer effects of the novel biguanide derivative IM176 with those of metformin and phenformin.

Methods

Prostate cancer cell lines and patient-derived castration-resistant prostate cancer (CRPC) cells were treated with IMI76, metformin, and phenformin. The effects of these agents on cell viability, annexin V-FITC apoptosis, mammalian target of rapamycin inhibition, protein expression and phosphorylation, and gene expression were evaluated.

Results

IM176 dose dependently reduced the viability of all prostate cancer cell lines tested, with IC50s (LNCaP: 18.5 μM; 22Rv1: 36.8 μM) lower than those of metformin and phenformin. IM176 activated AMP-activated protein kinase, inhibiting mammalian target of rapamycin and reducing the phosphorylation of p70S6K1 and S6. IM176 inhibited the expression of androgen receptor, the androgen receptor splice variant 7, and prostate-specific antigen in LNCaP and 22Rv1 cells. IM176 increased caspase-3 cleavage and annexin V-positive/propidium iodide–positive cells, which indicated apoptosis. Moreover, IM176 reduced viability, with low IC50, in cultured cells derived from two patients with CRPC.

Conclusion

The antitumor effects of IM176 were comparable with those of other biguanides. IM176 may therefore be a novel candidate for the treatment of patients with prostate cancer, including those with CRPC.

Keywords: Androgen receptor splice variant, Biguanide, Castration-resistant prostate cancer, Cell death

1. Introduction

Prostate cancer (PCa) can progress despite the depletion of serum testosterone. In these patients, an androgen receptor (AR)-related pathway is regarded as a central driver of disease progression.1,2 Despite treatment with recently developed hormonal agents, the median survival of patients with castration-resistant prostate cancer (CRPC) has been reported to be less than 2 years.2, 3, 4 Although multiple mechanisms have been shown to contribute to CRPC progression, AR splice variants (AR-Vs) are considered one of the most important mechanisms,5, 6, 7, 8 with one of these variants, AR-V7, considered a treatment selection biomarker in patients with CRPC.7,9,10 Because CRPC patients with AR-V7 showed unfavorable oncological outcomes after treatment with next-generation hormonal agents,11,12 novel drugs are still needed for treatment of patients with CRPC, particularly those with AR-V7.

Metformin, a biguanide derivative, is one of the most commonly used oral antidiabetic medications. Metformin has also been found effective against several types of cancer,13 as it interferes with the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathways.14,15 Metformin has potential as an anticancer drug for PCa,16,17 with several currently ongoing clinical trials evaluating metformin's role as an anticancer agent.18 Metformin has been shown to inhibit cancer cell proliferation and improve metabolic syndrome in PCa patients receiving androgen deprivation therapy.19

However, a recent meta-analysis showed that metformin did not improve disease-specific survival in patients with PCa20,21 and did not reduce the risk of PCa.22 Other biguanide derivatives with higher potency may show desired oncological outcomes in patients with PCa, particularly those with CRPC. The present study therefore evaluated the antitumor effects and mechanisms of action of IM176, a novel biguanide derivative, in PCa cell lines in cells derived from patients with CRPC. This study also compared the efficacy of IM176 with that of other biguanide derivatives, including metformin and phenformin.

2. Material and Methods

2.1. Reagents and antibodies

IM176 and phenformin were obtained from ImmunoMet Therapeutics Inc. (Houston, TX, USA), and metformin hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against phosphor-AMPK threonine 172 (pAMPK), AMPK, phospho-mTOR serine 2448 (pmTOR), mTOR, phospho-p70S6 kinase 1 threonine 389 (pp70S6K1), p70S6K1, phospho-S6 serine 235/236 (pS6), S6 caspase-3, and poly (ADP-ribose) polymerase were obtained from Cell Signaling Technology (Danvers, MA, USA); primary antibodies against AR, prostate-specific antigen (PSA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Santa Cruz Biotechnology (Dallas, TX, USA), and primary antibody against AR-V7 was from Precision Antibody (Columbia, MD, USA).

2.2. Cell culture

Human PCa cell lines, including PC3, DU145, LNCaP, 22Rv1, and VCaP, were obtained from the American Type Culture Collection (Manassas, VA, USA), maintained in Roswell Park Memorial Institute 1640 medium (PC3, DU145, LNCaP, 22Rv1), keratinocyte complete medium (0.05 mg/mL BPE, 5 ng/mL EGF, RWPE-1) or Dulbecco's modified Eagle's medium (VCaP), supplemented with 5%–10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin; and cultured in a 5% CO2 atmosphere at 37°C. Cells were used at passages 8–20. PCa tissue samples were obtained from patients who underwent transurethral resection of the prostate to relieve bladder outlet obstruction and were subsequently diagnosed with PCa at Asan Medical Center according to the protocol of the Institutional Review Board of Asan Medical Center. Tumor specimens were minced with scissors and digested by incubation in Roswell Park Memorial Institute 1640 containing 1 mg/mL type I collagenase (Sigma) for 1 hour at 37°C. Cells were washed with medium containing 10% FBS to inactivate collagenase and then with phosphate-buffered saline (PBS) to remove FBS. These cells were plated and maintained in Human Prostate Epithelial Cell Growth Medium (Lonza) in a 5% CO2 atmosphere at 37°C.

2.3. Cell viability assay

The effects of biguanide derivates on the viability of AR-expressing PCa cell lines and cells derived from patients with CRPC were evaluated using CellTiter-Glo Luminescent Cell viability Assays (Promega; Madison, WI, USA). The percentage of surviving cells was reported as the mean ± standard deviation of at least three replicates. In addition, the half maximal inhibitory concentration (IC50) of each drug in each cell line was determined with Prism version 8.0 software and expressed as microgram per milliliter.

2.4. Western blot analysis

Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Sigma). The cell lysates were microcentrifuged at 12,000×g for 5 min, and the supernatants were stored at 4°C. Protein concentrations were measured using the Bradford protein assay (Bio-Rad; Hercules, CA, USA). Equal amounts of proteins (20 μg in 20 μL) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride (Merck Millipore Ltd; Bedford, MA, USA) membranes. After blocking with 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C with shaking (100 rpm), followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized on X-ray film using Immobilon Western enhanced chemiluminescent solution (Millipore Corp). The expression of each protein was normalized to that of GAPDH in the same sample.

2.5. Real-time quantitative reverse transcription-polymerase chain reaction

Cells were seeded in 6-well plates (5 × 105 cells/well) containing 10% FBS for 18–24 h and incubated with IM176, metformin, or phenformin for 72 h. Total RNA was extracted from these cells, and 2 μg aliquots were reverse-transcribed using the first-strand complementary DNA synthesis kits (Toyobo; Osaka, Japan). The resulting complementary DNA samples were subjected to real-time polymerase chain reaction in an ABI 7500 sequence detector system using SYBR Green PCR Master Mix (Toyobo). Polymerase chain reaction amplification efficiency and linearity of each gene, including the target and control genes, were evaluated. The results were normalized to that of GAPDH messenger RNA (mRNA) and quantitatively analyzed using the 2 −ΔΔCt method. The amplification protocol consisted of an initial denaturation at 95°C for 20 seconds, followed by 40 cycles of denaturation at 95°C for 3 seconds, and annealing and extension at 60°C for 30 seconds. The melt curve stage consisted of 95°C for 15 seconds, a melt from 60°C for 1 minute to 95°C for 15 seconds with a ramp rate of 1%, and 60°C for 15 seconds. Amplification primers included those for AR: 5ʹ-CAGTGGATGGGCTGAAAAAT-3ʹ (forward) and 5ʹ-AAGCGTCTTGAGCAGGATGT-3ʹ (reverse); PSA: 5ʹ-CATCAGGAACAAAAGCGTGA-3ʹ (forward) and 5ʹ-ATATCGTAGAGCGGGTGTGG-3ʹ (reverse); and GAPDH: 5ʹ-CAATGACCCCTTCATTGACC-3ʹ (forward) and 5ʹ-GACAAGCTTCCCGTTCTCAG-3ʹ (reverse).

2.6. Annexin V staining apoptosis assay

Cells were seeded in 6-well plates (5 × 105 cells/well) containing 10% FBS for 18–24 h. After treatment with IM176 for 72 h, apoptotic and necrotic cell death was assessed by flow cytometry using the annexin V-FITC apoptosis detection kit (BD Biosciences; Bedford, MA, USA), according to the manufacturer's instructions. In these assays, cells positive for annexin V and those positive for both annexin V and propidium iodide (PI) represent the early and late apoptotic populations, respectively, whereas cells positive for PI only represent the necrotic population.

2.7. Immunofluorescence staining and confocal microscopy

Cells incubated with IM176, phenformin, or metformin were fixed with 4% paraformaldehyde in PBS for 30 minutes and permeabilized with 0.1% Triton X-100 in PBS for 30 minutes. These cells were incubated overnight at 4°C with primary antibodies against AR, pAMPK, and pS6 and then with secondary antibodies conjugated to fluorescent dye (Molecular Probes). The samples were mounted in VECTASHIELD containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and viewed by fluorescence microscopy (Olympus).

2.8. Statistical analysis

All data represent the mean ± standard deviation of at least three independent experiments. The results were compared statistically by using one-way analysis of variance, with p-values <0.05 considered statistically significant.

3. Results

3.1. Cell viability after treatment with IM176, phenformin, and metformin

Assessments of the expression of full-length androgen receptor (AR-FL) and AR splice variants (AR-Vs) in PCa cell lines showed that AR-FL was present in the LNCaP, 22Rv1, and VCaP PCa cell lines, whereas AR-Vs, including AR-V7, were expressed only in 22Rv1 and VCaP cells (Fig. 1a). Although treatment of PCa cell lines harboring AR with the biguanide derivatives IM176, phenformin, and metformin showed that all three drugs reduced the viability of LNCaP, 22Rv1, and VCaP cells, IM176 showed comparable effect at the lowest concentration (Fig. 1b). The IC50 values of IM176, phenformin, and metformin were 18.5 μmol/L, 38 μmol/L, and 4 mmol/L, respectively, in LNCaP cells; 36.8 μmol/L, 382.4 μmol/L, and 10 mmol/L, respectively, in 22Rv1 cells; and 21.7 μmol/L, 120.9 μmol/L, and 2.2 mmol/L, respectively, in VCaP cells.

Fig. 1.

Figure 1

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Effects of IM176, phenformin, and metformin on the viability of prostate cell lines harboring androgen receptor (AR) and AR splice variants (AR-Vs). (a) Western blots showing the level of AR in prostate cancer cell lines (PC3, DU145, LNCaP, 22Rv1, and VCaP). AR-Vs were identified with an antibody directed against the N-terminal site of AR, whereas AR splice variant 7 (AR-V7) was detected with a specific antibody directed against cryptic exon 3 sites in AR-V7. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (b) LNCaP, 22Rv1, and VCaP prostate cancer cells were treated with 0–100 μmol/L IM176, 0–1000 μmol/L phenformin, or 0–10 mmol/L metformin for 72 hours. Cell viability was determined by CellTiter-Glo. The results are expressed as the mean ± standard deviation of three independent experiments.

3.2. Activation of AMPK pathway and AR signaling by IM176, phenformin, and metformin

Treatment of LNCaP and 22Rv1 cells with the three biguanide derivatives IM176, phenformin, and metformin dose dependently increased the phosphorylation of AMPK at Thr172, although the level of AMPK was not changed (Fig. 2). Evaluation of downstream signaling showed that these three biguanide derivatives dose dependently reduced the phosphorylation of mTOR at serine 2448, of p70S6K1 at threonine 389, and of S6 at serine 235/236, although they had no effect on total mTOR and p70S6K1 expression. IM176 more effectively inhibited the mTOR pathway in LNCaP and 22Rv1 cells than phenformin and metformin.

Fig. 2.

Figure 2

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Effects of IM176, phenformin, and metformin on the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. (a–c) LNCaP and 22Rv1 cells were treated with the indicated concentrations of IM176 (a), phenformin (b), or metformin (c) for 24 hours. Western blot analysis was performed using antibodies against phospho-AMPK threonine 172 (pAMPK), AMPK, phospho-mTOR serine 2448 (pmTOR), mTOR, phospho-p70S6 kinase 1 threonine 389 (pp70S6K1), p70S6K1, phospho-S6 serine 235/236 (pS6), and S6. GAPDH was used as a loading control.

Compared with untreated cells, treatment with the IC50 concentrations of IM176, phenformin, and metformin markedly reduced the levels of AR mRNA to 10.11%, 12.37%, and 7.78%, respectively, in LNCaP cells and to 31.70%, 29.08%, and 37.72%, respectively, in 22Rv1 cells (Fig. 3a). Moreover, treatment with these concentrations of IM176, phenformin, and metformin reduced the levels of AR-V7 mRNA to 55.48%, 76.56%, and 58.03%, respectively, in 22Rv1 cells. Western blotting showed that all three biguanide derivatives reduced the levels of AR and PSA protein in LNCaP and 22Rv1 cells in a concentration-dependent manner (Fig. 3b), as well as reducing the levels of AR-Vs, including AR-V7, in 22Rv1 cells. In addition, IM176, phenformin, and metformin reduced cytosolic and nuclear ARs in both the LNCaP and 22Rv1 cell lines (Fig. 3c). Taken together, these results indicated that the biguanide derivatives effectively inhibited mTOR and AR signaling in PCa cells.

Fig. 3.

Figure 3

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Effects of IM176, phenformin, and metformin on the inhibition of AR, AR-V7, and prostate-specific antigen (PSA) expression. (a) LNCaP and 22Rv1 cells were treated with IC50 concentrations of IM176 (20 μmol/L and 40 μmol/L, respectively), phenformin (40 μmol/L and 400 μmol/L, respectively), and metformin (4 mmol/L and 10 mmol/L, respectively) for 72 hours, and relative mRNA levels of AR and AR-V7 were quantified by real-time PCR. Each point represents the mean ± standard deviation of triplicate determinations. ∗p < 0.05 compared with untreated control. (b) LNCaP and 22Rv1 cells were treated with the indicated concentrations of IM176, phenformin, and metformin for 72 hours. Western blot analysis was performed using antibodies to AR, AR-V7, and PSA, with GAPDH used as a loading control. (c) LNCaP and 22Rv1 cells were treated for 72 hours with the IC50 concentrations of IM176, phenformin, and metformin, as shown in (a), above. Samples were stained with antibody to AR (red) and the nuclear stain DAPI (blue). Images were captured on a fluorescence microscope. Scale bar, 50 μM. AR, androgen receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; PCR, polymerase chain reaction.

3.3. Cell death induced by IM176

Treatment with IM176 increased the level of cleaved caspase-3 in both LNCaP and 22Rv1 cells, although it had no effect on the level of pro-caspase 3 (Fig. 4a). In addition, IM176 increased the cleavage of poly (ADP-ribose) polymerase 1 in both cell lines. The rate of apoptosis was determined by flow cytometry of cells stained with annexin and PI (Fig. 4b). The percentages of LNCaP cells in early and late apoptosis were increased from 1.7% and 3%, respectively, in untreated cells to 1.8% and 37.9%, respectively, in IM176-treated cells. Similarly, the percentages of 22Rv1 cells in early and late apoptosis were increased from 0.9% and 7.2%, respectively, in untreated cells to 2.7% and 31.9%, respectively, in IM176-treated cells.

Fig. 4.

Figure 4

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Cell death induced by IM176 in LNCaP and 22Rv1 cell lines. (a and b) LNCaP and 22Rv1 cells were treated with the IC50 concentrations of IM176 (20 μmol/L and 40 μmol/L, respectively) for 72 hours, followed by Western blot analysis with antibodies against poly (ADP-ribose) polymerase (PARP)-1 and caspase-3. GAPDH was used as the loading control. (a) Flow cytometry analysis following annexin V/PI staining method. In this study, cells positive for annexin V (bottom right quadrant) and for both annexin V and propidium iodide (PI; upper right quadrant) represent early and late apoptotic populations, respectively, whereas cells positive for PI alone (upper left quadrant) represent the necrotic population (b). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

3.4. Effect of IM176 on patient-derived CRPC cells

Treatment of primary cultures of tumors from patients with CRPC with the three biguanide derivative drugs also reduced their viability, with IM176 showed a comparable effect at the lowest concentration (Fig. 5a). The IC50 values of IM176, phenformin, and metformin were 82.7 μmol/L, 466 μmol/L, and 12 mmol/L, respectively, for cells from CRPC patient number 1 (CRPC-P1); and 35 μmol/L, 1074 μmol/L, and 347.7 mmol/L, respectively, for cells from CRPC-P2. In addition, treatment of cells from CRPC-P1 with IM176 reduced the levels of cytosolic and nuclear AR (Fig. 5b), significantly reduced the level of pS6 (Ser235/236), and increased the level of pAMPK (Thr172). The IM176-induced reduction of AR and AR-Vs in primary CRPC-P1 cells was confirmed by Western blotting. Taken together, these results indicated that IM176 effectively induced cell death in cells from patients with CRPC.

Fig. 5.

Figure 5

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Anticancer effect of IM176 in primary cultures of cells from patients with castration-resistant prostate cancer (CRPC). (a) Primary cultures from two CRPC patients (CRPC-P1 and CRPC-P2) were treated with IM176 (0–100 μmol/L), phenformin (0–1000 μmol/L), and metformin (0–10 mmol/L) for 72 hours. Cell viability was determined by CellTiter-Glo. The results are expressed as the mean ± standard deviation of three independent experiments. (b) CRPC-P1 cells were treated with 100 μmol/L IM176 for 72 hours. Immunocytochemistry was performed using antibodies against AR, pAMPK, and pS6 (red), and the nuclei were stained with DAPI (blue). Images were captured on a fluorescence microscope. Scale bar, 50 μM. (c) CRPC-P1 cells were treated with 100 μmol/L IM176 for 72 hours, followed by Western blot analysis using anti-AR antibody. GAPDH was used as a loading control.

4. Discussion

Although several new treatments have been recently approved, the oncological outcomes of patients with CRPC are still suboptimal.3,4,23 In addition, CRPC patients with AR-V7 showed poorer oncological outcomes after treatment with next-generation hormonal therapy than patients without AR-V7.11,24 Despite drug-induced toxicities, taxane-based chemotherapy should be considered in CRPC patients with AR-V725 because AR-V7-expressing tumors were found to be sensitive to taxane-based chemotherapeutic agents.26, 27, 28 Thus, AR-V7 may be a potential biomarker for treatment selection in patients with CRPC.29,30 To date, however, there is no standard test for the presence of AR-V7. Thus, a novel agent that is effective in CRPC expressing AR-V7 may be a treatment option in most patients with CRPC, regardless of their AR-V7 status.

This study showed that IM176, a novel biguanide derivative, had antitumor effects, comparable with those of metformin and phenformin, in both hormone-sensitive and hormone-insensitive PCa cell lines. In addition, the IC50 of IM176 was lower than that of the other biguanide derivatives tested. The IC50 value of metformin in 22Rv1 cells was shown to be 1290 μg/mL, which was much higher than its therapeutic range and higher than its serum concentration in patients with renal impairment.31 Moreover, metformin-induced lactic acidosis, which occurs at concentrations >50 μg/mL, has been reported to have a 38% mortality rate.32 Although the IC50 of phenformin in 22Rv1 cells was lower than that of metformin, phenformin-associated lactic acidosis has been reported at concentrations ranging from 20 to 625 ng/mL, similar to its IC50 in 22Rv1 cells. Thus, neither metformin nor phenformin could be used clinically to treat patients with PCa. However, the IC50 of IM176 in 22Rv1 cells was much lower than that of either metformin or phenformin, with similar results observed in primary cells from patients with CRPC. Thus, IM176 may be clinically acceptable for the treatment of these patients, although additional in vitro and in vivo studies are required.

As predicted, the mechanism underlying the antineoplastic effects of IM176 was similar to that of the other biguanide derivatives. Metformin has shown potential antineoplastic effects in several types of cancer,33,34 as well as showing synergistic effects when added to other anticancer drugs.35 The antineoplastic effects of metformin have been attributed primarily to AMPK activation and mTOR inhibition.36 In the current study, IM176 showed similar effects on the AMPK-mTOR pathway, resulting in tumor cell death.

The present study also found that the three biguanide drugs reduced AR and AR-V7 expression, in agreement with previous findings. Although attempts have been made to determine the relationship between the AMPK-mTOR and AR signaling pathways,37 this relationship has not been verified. The present study showed that PSA was regulated by the AMPK-mTOR pathway, independently of AR signaling. Phosphorylation of S6 kinase, which acts downstream of p70S6K1, has been shown to regulate AR function and PCa cell growth.38 In addition, IM176 suppressed AR and AR-V7 expression, which may regulate the AMPK-mTOR pathway because phosphorylation of AMPK was activated by AR knockdown. We also showed that IM176 had antitumor effects via AMPK-mediated inhibition of the mTOR pathway and cell death in AR-deficient PCa (Supplementary Figure 1). However, the relationship between the AMPK-mTOR and AR signaling pathways is quite complex, with additional studies required to determine this relationship.

Because metformin is both safe and cost-effective, it has been used in most studies of the anticancer effects of biguanide derivatives. Although a recent prospective randomized clinical trial tested the therapeutic effects of hormonal therapy plus metformin in men with advanced PCa, the effects of metformin on PCa-specific mortality remain unclear. Biguanide derivative drugs with higher potency than metformin may be needed to achieve desired oncological outcomes in patients with PCa. IM176, in combination with conventional and next-generation hormonal therapeutic agents, maytlsb therefore be a potential candidate for the treatment of CRPC.

The present study had several limitations. First, this study did not assess the in vivo anticancer effects of IM176, whether in animal models or in patients with PCa. In addition, the toxicity profile and kinetics of IM176 remain to be determined, although they may be similar to those of the other biguanides. Moreover, the synergistic effects of combination treatment with IM176 and currently available drugs, including next-generation hormonal agents and/or taxane-based chemotherapeutic agents, need to be determined.

5. Conclusions

IM176 showed antitumor effects, comparable with those of metformin and phenformin, via the AMPK-mediated inhibition of the mTOR pathway and the AR signaling pathway. IM176 had lower IC50 values than metformin and phenformin in PCa cell lines, including in androgen-insensitive PCa cell lines. IM176 may therefore be a novel candidate for the treatment of patients with PCa, including CRPC.

Conflict of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by a grant from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea (grant number: 2018-614).

Acknowledgement

The authors thank ImmunoMet Therapeutics Inc. for providing IM176. ImmunoMet Therapeutics Inc. was not involved in the study design, interpretation of data, or writing of the article. The authors would like to thank Bioedit LTD (www.bioedit.co.uk) for the English language review.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.prnil.2022.11.003.

Contributor Information

Dalsan You, Email: dalsanyou@amc.seoul.kr.

Jung Jin Hwang, Email: jjhwang@amc.seoul.kr.

Choung-Soo Kim, Email: cskim37345806@gmail.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Supplementary Figure 1.

Supplementary Figure 1

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Anticancer effect of IM176 in AR-deficient PCa cells. (a) PC3 and DU145 prostate cancer cells were treated with 0–100 μmol/L IM176, 0–1000 μmol/L phenformin, or 0–10 mmol/L metformin for 72 hours. Cell viability was determined by CellTiter-Glo. The results are expressed as the mean ± SD of three independent experiments. The IC50 values of IM176, phenformin, and metformin were 28.8 μmol/L, 254.1 μmol/L, and 8.6 mmol/L, respectively, in PC3 cells; and 38.88 μmol/L, 492.3 μmol/L, and 8.4 mmol/L, respectively, in DU145 cells. (b) PC3 cells were treated with the indicated concentrations of IM176 for 24 hours. Western blot analysis was performed using antibodies against phospho-AMPK threonine 172 (pAMPK), AMPK, phospho-mTOR serine 2448 (pmTOR), mTOR, phospho-p70S6 kinase 1 threonine 389 (pp70S6K1), p70S6K1, phospho-S6 serine 235/236 (pS6), and S6. GAPDH was used as a loading control. (c) PC3 cells were treated with the IC50 concentrations of IM176 (40 μmol/L, respectively) for 72 hours, followed by Western blot analysis with antibodies against poly (ADP-ribose) polymerase (PARP)-1, caspase-3, and cleaved caspase-3. GAPDH was used as the loading control

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