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A Quinone-Containing Compound Enhances Camptothecin-Induced Apoptosis of Lung Cancer Through Modulating Endogenous ROS and ERK Signaling

Abstract

The natural compound camptothecin (CPT) derivatives have widely been used for anti-cancer treatments, including lung cancer. However, many chemoresistant cancer cells often develop a relatively higher threshold for inducing apoptosis, causing a limited efficacy of anti-cancer drugs. Likewise, lung cancer cells acquire chemoresistance against CPT analogs, such as irinotecan and topotecan, finally resulting in an unsatisfied outcome and poor prognosis of lung cancer patients. TFPP is a quinone-containing compound as a candidate for CPT-based combination chemotherapy. In this study, we examined the effect of TFPP and CPT cotreatment on non-small cell lung cancer (NSCLC) cells. Cell proliferation and flow cytometry-based Annexin-V/PI staining assays demonstrated the synergistic effect of TFPP on CPT-induced apoptosis in both NSCLC A549 and H1299 cells. The results of CPT and TFPP cotreatment cause the regulation of the ERK-Bim axis and the activation of mitochondrial-mediated caspase cascade, including caspase-9 and caspase-3. Besides, TFPP significantly enhanced CPT-induced endogenous reactive oxygen species (ROS) in the two NSCLC cells. In contrast, the treatment of N-acetyl-l-cysteine (NAC), an ROS scavenger, rescues the apoptosis of NSCLC cells induced by TFPP and CPT cotreatment, suggesting that the synergistic effect of TFPP on CPT-induced anti-NSCLC cells is through upregulating ROS production. Consequently, our results suggest that TFPP sensitizes NSCLC towards CPT-based chemotherapy may act through decreasing the apoptosis-initiating threshold. Therefore, TFPP may be a promising chemosensitizer for lung cancer treatment, and the underlying mechanism warrants further.

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Acknowledgments

This study was supported by the Grants #MOST101-2320-B-037-046-MY3 and #MOST 102-2632-B-037-001-MY3 from the Ministry of Science and Technology (MOST), Taiwan; by the Grant #KMU-TP104A03 from the KMU Research Project, Taiwan; by the Grant #NSYSUKMU104-P031 from the NSYSU-KMU Joint Research Project, Taiwan; and by the Grant #104-CM-KMU-006 from ChiMei-KMU Joint Research Project, Taiwan. We are also grateful for the instrument supports of flow cytometry from the Center for Research Resources and Development at Kaohsiung Medical University, Taiwan.

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Correspondence to Hurng-Wern Huang or Chien-Chih Chiu.

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Supplementary Fig. l

The synergistic effects of CPT and TFPP combination on anti-proliferation of NSCLC cells using the Chou–Talalay method (Chou and Talalay 1981, 1984). H1299 cells were treated with the indicated concentrations of CPT and TFPP combinations for 48 h, respectively. The proliferative rate of H1299 cells was measured by Trypan blue exclusion assay. (A) The synergistic effects of CPT and TFPP combination are evaluated using the program CalcuSyn and the lines represent the effective doses (EDs) of the effects of the two compounds to achieve 50, 75, or 90 % inhibition. The dots represent the experimental doses used for achieving the above inhibitory effects. (B) The table represent the mean values of combination index, which represent the degree of CPT and TFPP interactions, < 1 for synergistic-, = 1 for synergistic-, and > 1 for antagonistic effects, respectively. Data are presented as mean ± SD from at least three independent experiments (TIFF 191 kb)

Supplementary Fig.

 2 The results of MitoSox assay. (A) A549 and H1299 cells were pre-treated with either 2 mM ROS scavenger NAC or ERK inhibitor PD98059 for 3 h, and were then subject to CPT treatment for 48 h. Afterward, the cells were staining with a ROS-sensitive dye MitoSox, which is mitochondria-selective. (D) The quantificative analysis of (B). Data are presented as mean ± SD (anot statistical significance; b p < 0.05; c p < 0.001) (TIFF 275 kb)

Supplementary Fig.

 3 Determining the role of MAPK in CPT-induced apoptosis of NSCLC cells. (A) A549 and H1299 cells were pre-treated with ROS scavenger NAC or MAPK specific inhibitors, and were then subject to CPT treatment for 48 h, respectively. Afterward, the apoptosis was detected using a flow cytometry-based Annexin-V-staining assay. (B) The quantitative analysis of (A). Data are presented as mean ± SD (anot statistical significance; b p < 0.05; c p < 0.001) (TIFF 450 kb)

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Chou, HL., Fong, Y., Wei, CK. et al. A Quinone-Containing Compound Enhances Camptothecin-Induced Apoptosis of Lung Cancer Through Modulating Endogenous ROS and ERK Signaling. Arch. Immunol. Ther. Exp. 65, 241–252 (2017). https://doi.org/10.1007/s00005-016-0424-8

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Keywords

  • Non-small cell lung cancer
  • Chemoresistance
  • Apoptosis threshold
  • Camptothecin
  • TFPP
  • ERK signaling pathway