Korean J Physiol Pharmacol.  2025 May;29(3):321-335. 10.4196/kjpp.24.274.

Enhancing doxorubicin’s anticancer impact in colorectal cancer by targeting the Akt/Gsk3β/mTOR-SREBP1 signaling axis with an HDAC inhibitor

• Zhao H
• Wu Y
• Kim SM

• KMID: 2567190
• DOI: http://doi.org/10.4196/kjpp.24.274

Abstract

Colorectal cancer ranks third in global incidence and is the second leading cause of cancer-related mortality. Doxorubicin, an anthracycline chemotherapeutic drug, is integral to current cancer treatment protocols. However, toxicity and resistance to doxorubicin poses a significant challenge to effective therapy. Panobinostat has emerged as a critical agent in colorectal cancer treatment due to its potential to overcome doxorubicin resistance and enhance the efficacy of existing therapeutic protocols. This study aimed to evaluate the capability of panobinostat to surmount doxorubicin toxicity and resistance in colorectal cancer. Specifically, we assessed the efficacy of panobinostat in enhancing the therapeutic response to doxorubicin in colorectal cancer cells and explored the potential synergistic effects of their combined treatment. Our results demonstrate that the combination treatment significantly reduces cell viability and colony-forming ability in colorectal cancer cells compared to individual treatments. The combination induces significant apoptosis, as evidenced by increased levels of cleaved PARP and cleaved caspase-9, while also resulting in a greater reduction in p-Akt/p-GSK-3β/mTOR expression, along with substantial decreases in c-Myc and SREBP-1 levels, compared to monotherapies. Consistent with the in vitro experimental results, the combination treatment significantly inhibited tumor formation in colorectal cancer xenograft nude mice compared to the groups treated with either agent alone. In conclusion, our research suggests that the panobinostat effectively enhances the effect of doxorubicin and combination of two drugs significantly reduced colorectal cancer tumor growth by targeting the Akt/ GSK-3β/mTOR signaling pathway, indicating a synergistic therapeutic potential of these two drugs in colorectal cancer treatment.

Keyword

c-Myc; Colorectal neoplasms; Doxorubicin; Panobinostat; Proto-Oncogene Proteins c-akt/ glycogen synthase kinase-3 beta/ mammalian target of rapamycin; Sterol regulatory element binding protein 1

Figure

• 

  Fig. 1 The combination treatment of PAN and DOX inhibited cell proliferation and colony formation in HCT116 and SW480 CRC cells. (A) SW480 and HCT116 cells were treated with 5 nM PAN, 0.5 µM DOX, or the combination. Cell viability assays were used to evaluate cell proliferation. (B) Colony formation assay. The combination treatment of PAN and DOX significantly inhibited colony formation of HCT116 and SW480 cells compared to the single agents alone. Data represent the mean ± SEM of three separate, triplicate-dish tests. CRC, colorectal cancer; DMSO, dimethyl sulfoxide; CONT, control; PAN, panobinostat; DOX, doxorubicin. **p < 0.01 compared to the control group; ##p < 0.01 compared to the combination group.

• 

  Fig. 2 Effect of PAN and DOX on the apoptosis of HCT116 and SW480 CRC cells. (A) The CRC cells were collected to assess the apoptotic rate using FITC/PI double positive staining following treatment with PAN, DOX, or the combination. (B) The proportion of Sub-G1 increased after treatment with PAN, DOX, or the combination. (C) Following treatment of CRC cells with PAN, DOX, or the combination for 48 h, apoptosis-associated proteins c-PARP, PARP, c-Caspase 9, and Caspase 9 expression were semi-quantified by Western blotting. Values were normalized to GAPDH. Data represent the mean ± SEM of three separate, triplicate-dish tests. CRC, colorectal cancer; CONT, control; PAN, panobinostat; DOX, doxorubicin; PI, propidium iodide; PARP, poly (ADP-ribose) polymerase 1; c, cleaved; n.s., not significant. *p < 0.05 and **p < 0.01 compared to the control group; #p < 0.05 and ##p < 0.01 compared to the combination group.

• 

  Fig. 3 Effects of PAN and DOX on migration of HCT116 and SW480 CRC cells. (A) The migration rate of CRC cells was assessed by wound healing assay following treatment with PAN, DOX, or the combination treatment. The cell migration ability was significantly inhibited when treated with PAN and DOX at 24 h and 48 h. (B) Migration-related protein expression levels of E-cadherin, uPA, and MMP-9 were semi-quantified by Western blotting following treatment with PAN, DOX, or the combination treatment. Data are the mean ± SEM of more than three independent experiments with triplicate dishes. CRC, colorectal cancer; CONT, control; PAN, panobinostat; DOX, doxorubicin; MMP-9, matrix metalloproteinase-9; uPA, urokinase-type plasminogen activator; n.s., not significant; *p < 0.05 and **p < 0.01 compared to the control group; #p < 0.05 and ##p < 0.01 compared to the combination group.

• 

  Fig. 4 Effects of PAN and DOX on Akt signaling. Protein expression levels of Akt, p-Akt, Gsk3β, and p-Gsk3β were assessed using Western blotting following treatment with PAN, DOX, or the combination for 48 h. Values were normalized to GAPDH. Data represent the mean ± SEM of three separate, triplicate-dish tests. CONT, control; PAN, panobinostat; DOX, doxorubicin; p, phosphorylated; Gsk3β, glycogen synthase kinase 3β; n.s., not significant. *p < 0.05 and **p < 0.01 compared to the control group; ##p < 0.01 compared to the combination group.

• 

  Fig. 5 Effects of PAN and DOX on the Akt/mTOR signaling pathway following activation or inhibition of Akt expression. (A) Protein expression levels of mTOR, p-mTOR, p70S6K, and p-p70S6K were assessed using Western blotting following treatment with PAN, DOX, or the combination. (B) Protein expression levels of c-Myc and SREBP-1 were assessed using Western blotting following treatment with PAN, DOX, or the combination. (C) Protein expression levels of Akt signaling pathway and downstream proteins were assessed using Western blotting following treatment with an activator (SC79) or inhibitor (LY294002) of Akt, PAN, DOX, or the combination. Values were normalized to GAPDH. Data represent the mean ± SEM of three separate, triplicate-dish tests. CONT, control; PAN, panobinostat; DOX, doxorubicin; p, phosphorylated; mTOR, mammalian target of rapamycin; p70S6K, ribosomal protein S6 kinase beta-1; SREBP-1, sterol regulatory element-binding protein 1; n.s., not significant. *p < 0.05 and **p < 0.01 compared to the control group; #p < 0.05 and ##p < 0.01 compared to the combination group.

• 

  Fig. 6 In vivo effects of PAN and DOX on the tumor growth of HCT116 xenograft mouse model. (A) On day 0, a xenograft mouse model was established and treated with DMSO, PAN, DOX or PAN + DOX on days 6 through 26. On day 26, all mice were sacrificed and samples were harvested. (B) After therapy, the body weight of the mice with tumors was measured every two days. (C) Representative tumor images and (D) final tumor weight was obtained at the end of the experiment. (E) The tumor volume was monitored every two days. Values were normalized to GAPDH. (F) ALT, (G) AST, (H) BUN, and (I) creatinine levels were assessed. (J) The protein levels of Akt, p-Akt, p70S6K, p-p70S6K, c-Myc, and SREBP-1 from mouse tissue were measured. Three samples were selected randomly from each group. Values were normalized to GAPDH. Data represent the mean ± SEM of more than three separate, triplicate-dish tests. DMSO, dimethyl sulfoxide; CONT, control; PAN, panobinostat; DOX, doxorubicin; p, phosphorylated; mTOR, mammalian target of rapamycin; p70S6K, ribosomal protein S6 kinase beta-1; SREBP-1, sterol regulatory element-binding protein 1; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; n.s., not significant. *p < 0.05 and **p < 0.01 compared to the control group; #p < 0.05 and ##p < 0.01, compared to the combination group.

• 

  Fig. 7 Schematic diagram. The combination treatment of PAN and DOX inhibits cell growth and induces apoptosis of CRC cells by targeting the Akt signaling pathway. LY294002 further inhibits Akt activity and its downstream functions after treatment with the combination of PAN and DOX. CRC, colorectal cancer; PAN, panobinostat; DOX, doxorubicin; p, phosphorylated; Gsk3β, glycogen synthase kinase 3β; mTOR, mammalian target of rapamycin; P70S6K, ribosomal protein S6 kinase beta-1; SREBP-1, sterol regulatory element-binding protein 1; MMP-9, matrix metalloproteinase-9; uPA, urokinase-type plasminogen activator; PARP, poly (ADP-ribose) polymerase.

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