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SIRT3-Mediated SOD2 and PGC-1α Contribute to Chemoresistance in Colorectal Cancer Cells

  • Translational Research
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

Anticancer drugs generate excessive reactive oxygen species (ROS), which can cause cell death. Cancer cells can resist this oxidative stress, but the mechanism of resistance and associations with chemoresistance are unclear. Here, we focused on Sirtuin 3 (SIRT3), a deacetylating mitochondrial enzyme, in oxidative stress resistance in colorectal cancer (CRC).

Methods

To evaluate SIRT3-related changes in mitochondrial function, ROS (mtROS) induction, and apoptosis, we used the human CRC cell lines HT29 and HCT116 transfected with short-hairpin RNA targeting SIRT3 and small interfering RNAs targeting superoxide dismutase 2 mitochondrial (SOD2) and peroxisome proliferator–activated receptor γ coactivator-1 (PGC-1α). In 142 clinical specimens from patients with CRC, we also assessed the association of SIRT3 protein levels (high/low) and prognosis.

Results

SIRT3 expression correlated with mtROS generation and apoptosis induction in cells treated with anticancer agents. Suppressing SIRT3 increased mtROS levels and cell sensitivity to anticancer agents. SIRT3 knockdown decreased SOD2 expression and activity, and suppressing SOD2 also improved sensitivity to anticancer drugs. In addition, SIRT3 was recruited with PGC-1α under oxidative stress, and suppressing SIRT3 decreased PGC-1α expression and mitochondrial function. PGC-1α knockdown decreased mitochondrial activity and increased apoptosis in cells treated with anticancer drugs. In resected CRC specimens, high vs low SIRT3 protein levels were associated with significantly reduced cancer-specific survival.

Conclusions

SIRT3 expression affected CRC cell chemoresistance through SOD2 and PGC-1α regulation and was an independent prognostic factor in CRC. SIRT3 may be a novel target for CRC therapies and a predictive marker of sensitivity to chemotherapy.

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Author information

Authors and Affiliations

  1. Department of Gastroenterological Surgery, Osaka University, Suita, Osaka, Japan

    Masakatsu Paku MD, Shiki Fujino MD, PhD, Takayuki Ogino MD, PhD, Hidekazu Takahashi MD, PhD, Norikatsu Miyoshi MD, PhD, Mamoru Uemura MD, PhD, Tunekazu Mizushima MD, PhD, Hirofumi Yamamoto MD, PhD, Yuichiro Doki MD, PhD & Hidetoshi Eguchi MD, PhD

  2. Department of Gastroenterological Surgery, Osaka Prefectural Hospital Organization, Osaka International Cancer Institute, Chuo-ku, Osaka, Japan

    Naotsugu Haraguchi MD, PhD

  3. Department of Genomic Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

    Mitsunobu Takeda MD, PhD

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  1. Masakatsu Paku MD
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  2. Naotsugu Haraguchi MD, PhD
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Corresponding author

Correspondence to Naotsugu Haraguchi MD, PhD.

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Disclosure

Masakatsu Paku, Naotsugu Haraguchi, Mitsunobu Takeda, Shiki Fujino, Takayuki Ogino, Norikatsu Myoshi, Hidekazu Takahashi, Mamoru Uemura, Tsunekazu Mizushima, Hirofumi Yamamoto, Yuichiro Doki, and Hidetoshi Eguchi declare no competing financial interests.

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Supplemental Fig.

 1. (a, b) The level of mtROS and apoptosis was evaluated in human CRC cells with the treatment of anticancer agents. The positive rates of (a) MitoSOX Red and (b) Annexin V were assessed in each cell line exposed to 5-FU (10 μM), OXA (10 μM), and SN-38 (0.2 μM) for 48 h. CRC: colorectal cancer; mtROS: mitochondrial reactive oxygen species; 5-FU: 5-fluorouracil; OXA: oxaliplatin; SN-38: 7-ethyl-10-hydroxycamptothecin (TIFF 2197 kb)

Supplemental Fig.

 2. The sensitivity to 5-FU, OXA, and SN-38 in human CRC cells was assessed by CCK-8 assay. Relative absorbance at 450 nm for each anticancer drug tested at 10 dilutions. CRC: colorectal cancer; 5-FU: 5-fluorouracil; OXA: oxaliplatin; SN-38: 7-ethyl-10-hydroxycamptothecin; CCK-8: Cell Counting Kit-8 (TIFF 2197 kb)

Supplemental Fig.

 3. (a, b) The value of mtROS and apoptosis was assessed in the control cells and the shSIRT3-#2 cells with the treatment of anticancer agents. (a) The fluorescence intensities of MitoSOX Red and (b) Annexin V were higher in the shSIRT3-#2 cells exposed to 5-FU (10 μM), OXA (10 μM), or SN-38 (0.2 μM) for 48 h. (c) The sensitivity to H2O2 in the control cells and the shSIRT3-#2 cells was measured by CCK-8 assay. Relative absorbance at 450 nm for each anticancer agent tested at 10 dilutions in the control cells and the shSIRT3 cells. The shSIRT3-#2 cells were more sensitive to each agent than the control cells. CRC: colorectal cancer; mtROS: mitochondrial reactive oxygen species; 5-FU: 5-fluorouracil; OXA: oxaliplatin; SN-38: 7-ethyl-10-hydroxycamptothecin; CCK-8: Cell Counting Kit-8; shSIRT3: shRNA targeting SIRT3 (TIFF 2197 kb)

Supplemental Fig.

 4. (a) The fluorescence intensity of MitoTracker Green was lower in the shSIRT3-#2 cells exposed to 5-FU (10 μM), OXA (10 μM), or SN-38 (0.2 μM) for 48 h. (b, c) The values for mitochondrial activity and apoptosis were assessed in the control cells and the siPGC-1α cells with the treatment of anticancer agents. The fluorescence intensities of (a) MitoTracker Green and (b) Annexin V were lower in the siPGC-1α cells exposed to 5-FU (10 μM), OXA (10 μM), or SN-38 (0.2 μM) for 48 h. CRC: colorectal cancer; 5-FU: 5-fluorouracil; OXA: oxaliplatin; SN-38: 7-ethyl-10-hydroxycamptothecin; shSIRT3: shRNA targeting SIRT3; siPGC-1α: PGC-1α siRNA (TIFF 2197 kb)

Supplemental Fig.

 5. (a) The expression levels of SIRT3 were detected in the 13 human CRC frozen tissue samples from the 155 samples we collected and used for immunohistochemistry: weakly positive (n = 3), moderately positive (n = 4), strongly positive (n = 6). Weakly positive human CRC samples had low SIRT3 expressions, and SIRT3 expressions increased as the staining intensity increased. CRC: colorectal cancer (TIFF 2197 kb)

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Paku, M., Haraguchi, N., Takeda, M. et al. SIRT3-Mediated SOD2 and PGC-1α Contribute to Chemoresistance in Colorectal Cancer Cells. Ann Surg Oncol 28, 4720–4732 (2021). https://doi.org/10.1245/s10434-020-09373-x

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