Korean J Physiol Pharmacol.  2022 Sep;26(5):367-375. 10.4196/kjpp.2022.26.5.367.

Atorvastatin inhibits the proliferation of MKN45-derived gastric cancer stem cells in a mevalonate pathway-independent manner

• Choi YS
• Cho HJ
• Jung HJ

• KMID: 2532766
• DOI: http://doi.org/10.4196/kjpp.2022.26.5.367

Abstract

Gastric cancer stem cells (GCSCs) are a major cause of radioresistance and chemoresistance in gastric cancer (GC). Therefore, targeting GCSCs is regarded as a powerful strategy for the effective treatment of GC. Atorvastatin is a widely prescribed cholesterol-lowering drug that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme in the mevalonate pathway. The anticancer activity of atorvastatin, a repurposed drug, is being investigated; however, its therapeutic effect and molecular mechanism of action against GCSCs remain unknown. In this study, we evaluated the anticancer effects of atorvastatin on MKN45-derived GCSCs. Atorvastatin significantly inhibited the proliferative and tumorsphere-forming abilities of MKN45 GCSCs in a mevalonate pathway-independent manner. Atorvastatin induced cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade. Furthermore, atorvastatin exerted an antiproliferative effect against MKN45 GCSCs by inhibiting the expression of cancer stemness markers, such as CD133, CD44, integrin α6, aldehyde dehydrogenase 1A1, Oct4, Sox2, and Nanog, through the downregulation of β-catenin, signal transducer and activator of transcription 3, and protein kinase B activities. Additionally, the combined treatment of atorvastatin and sorafenib, a multi-kinase targeted anticancer drug, synergistically suppressed not only the proliferation and tumorsphere formation of MKN45 GCSCs but also the in vivo tumor growth in a chick chorioallantoic membrane model implanted with MKN45 GCSCs. These findings suggest that atorvastatin can therapeutically eliminate GCSCs.

Keyword

Atorvastatin; Cancer stem cells; Gastric cancer; Mevalonate; Sorafenib

Figure

• 

  Fig. 1 Atorvastatin inhibits the proliferation of MKN45 gastric cancer stem cells (GCSCs) in a mevalonate pathway-independent manner. (A) Expression levels of key GCSC markers in MKN45 adherent and tumorsphere cells during serial cell passaging (passages 1–3) under different culture conditions. The protein expression was analyzed by Western blotting. β-actin was used as a loading control. ***p < 0.001. Each value represents the mean ± SD from three independent experiments. (B) Effect of atorvastatin on the proliferation of MKN45 GCSCs. The cells were treated with atorvastatin (0–25 µM) for 7 days. Cell proliferation was measured using the CellTiter-Glo luminescent assay system. ***p < 0.001 vs. the control. (C, D) Effect of atorvastatin on the tumorsphere-forming ability of MKN45 GCSCs. The cells were treated with atorvastatin (0–25 µM) for 7 days. The number of tumorspheres was counted under an optical microscope (scale bar, 100 μm). ***p < 0.001 vs. the control. (E) Protein expression levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) in MKN45 adherent and tumorsphere cells. (F–H) Effect of atorvastatin on the proliferation (F) and tumorsphere-forming ability (G, H) of MKN45 GCSCs in the absence or presence of mevalonate. The cells were treated with atorvastatin and mevalonate for 7 days (scale bar, 100 μm). ***p < 0.001.

• 

  Fig. 2 Atorvastatin induces cell cycle arrest and apoptosis in MKN45 gastric cancer stem cells (GCSCs). (A–C) MKN45 GCSCs were treated with atorvastatin for 4 days. (A) The cell cycle distribution was evaluated using a Muse Cell Analyzer with Muse Cell Cycle kit following the manufacturer’s instructions. (B) Apoptotic cells were detected using a Muse Cell Analyzer with Muse Annexin V & Dead Cell kit following the manufacturer’s instructions. (C) The protein expression levels of the cell cycle and apoptosis regulators were detected by western blotting. β-actin was used as a loading control. PARP, poly (ADP‐ribose) polymerase; CDK4, cyclin-dependent kinase 4. **p < 0.01, ***p < 0.001 vs. the control.

• 

  Fig. 3 Atorvastatin downregulates the expression of cancer stemness markers and Wnt/β-catenin/signal transducer and activator of transcription 3 (STAT3)/protein kinase B (AKT) signaling pathways in MKN45 gastric cancer stem cells (GCSCs). (A, B) MKN45 GCSCs were treated with atorvastatin for 2 days. The protein expression levels were determined by western blotting. β-actin was used as an internal control. ALDH1A1, aldehyde dehydrogenase 1A1; NF-κB, nuclear factor-κB; GSK3β, glycogen synthase kinase 3β. **p < 0.01, ***p < 0.001 vs. the control.

• 

  Fig. 4 Atorvastatin increases the chemosensitivity of MKN45 gastric cancer stem cells (GCSCs) to sorafenib in vitro and in vivo. (A–C) MKN45 GCSCs were treated with the indicated concentrations of sorafenib and atorvastatin for 7 days. (A) Cell proliferation was measured using the CellTiter-Glo luminescent assay system. (B, C) The number of formed tumorspheres was counted under an optical microscope (scale bar, 100 μm). (D) MKN45 GCSCs were treated with the indicated concentrations of sorafenib and atorvastatin for 2 days. Cell cycle distribution was evaluated using a Muse Cell Analyzer with Muse Cell Cycle kit. (E) MKN45 GCSCs were mixed with Matrigel in the absence or presence of sorafenib (5 µg/egg) and atorvastatin (10 µg/egg) and implanted onto the chorioallantoic membrane (CAM) surface inside the silicone ring of fertilized chick eggs. After incubation for 7 days, the CAMs were photographed, the formed tumors were retrieved, and the tumor weight was calculated. NT, no treatment. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the compound alone.

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