Cytoplasmic SHMT2α promotes DNA damage repair and sustains cancer stemness to confer cisplatin resistance in non-small cell lung cancer: an in vitro study
Abstract
MethodsCytoplasmic and mitochondrial proteins were extracted from A549 and A549-DDP cells, and the expression levels of SHMT2 in the respective subcellular fractions were detected by Western blot. Stable cell lines overexpressing cytoplasmic SHMT2α or knocking out SHMT2 were established. Cell viability following treatment with gradient concentrations of DDP (0.0, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0 μmol/L) for 48 h was assessed using the cell counting kit-8 (CCK-8) assay. Western blot and immunofluorescence assays were employed to evaluate the alterations in the protein expression of γ-H2AX, a pivotal biomarker for DNA damage repair, in stably transfected cells after treatment with 5 μmol/L DDP for 48 h. The expression of cancer stem cell markers was examined by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and a tumor stem cell spheroid formation assay was used to evaluate stemness maintenance capacity. Data were analyzed using GraphPad Prism (v8.0), and all experiments were independently repeated three times.ResultsThe total SHMT2 protein expression levels were similar between A549 and A549-DDP cells (P=0.129 6); however, compared to A549 cells, SHMT2 expression was higher in the cytoplasm of DDP-resistant cells (P=0.033 7). Overexpression of cytoplasmic SHMT2α led to an increased half-maximal inhibitory concentration (IC₅₀)（A549: P=0.007 7; H1299: P<0.001） and decreased γ-H2AX expression (A549: P=0.013 6; H1299: P=0.024 2) following 5 μmol/L DDP for 48 h, whereas SHMT2 knockout resulted in a decreased IC₅₀ (A549: sg-SHMT2-1: P=0.010 1; sg-SHMT2-2: P=0.006 6; H1299: sg-SHMT2-1: P=0.029 0; sg-SHMT2-2: P=0.013 7) and increased γ-H2AX expression (A549: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.015 5; H1299: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.002 4). Furthermore, overexpression of the cytoplasmic SHMT2α promoted the expression of cancer stem cell markers (A549: SOX2, CD133, BMI1: P<0.001; OCT4: P=0.006 8; Nanog: P=0.002 8; CD44: P=0.043 5; H1299: OCT4, Nanog, SOX2, CD133, CD44: P<0.001; BMI1: P=0.003 4) and enhanced the tumorsphere formation efficiency of NSCLC cancer stem cells (A549: P=0.012 8; H1299: P<0.001). Conversely, SHMT2 knockout reduced the levels of cancer stem cell markers (A549: SOX2, CD133: P<0.001; OCT4: sg-SHMT2-1: P=0.018 4; sg-SHMT2-2: P=0.048 5; Nanog: sg-SHMT2-1: P=0.002 9; sg-SHMT2-2: P=0.002 8; BMI1: sg-SHMT2-1: P=0.004 5; sg-SHMT2-2: P=0.003 5; CD44: sg-SHMT2-1: P=0.003 8; sg-SHMT2-2: P=0.002 7; H1299: OCT4, Nanog, SOX2, CD133, BMI1: P<0.001; CD44: sg-SHMT2-1: P=0.004 0; sg-SHMT2-2: P=0.004 3) and suppressed sphere formation efficiency (A549: P<0.001; H1299: P<0.001).ConclusionSHMT2 is highly expressed in the cytoplasm of DDP-resistant non-small cell lung cancer cells. It reduces cellular sensitivity to DDP by promoting DNA damage repair and sustaining tumor cell stemness, thereby providing a potential therapeutic target for reversing DDP resistance in non-small cell lung cancer.Background and purposeThe incidence and mortality of lung cancer have remained persistently high. Non-small cell lung cancer (NSCLC), the most common histological type, presents cisplatin (DDP) resistance as one of the most challenging clinical hurdles. SHMT2, a key metabolic enzyme that catalyzes the conversion of serine to glycine with concomitant generation of one-carbon units, is localized in both mitochondria and the cytoplasm and is highly expressed in various malignancies. This study aimed to investigate the role and molecular mechanism of cytoplasmic SHMT2α in DDP resistance of NSCLC.
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BibTeX
@article{xinyu2026cytoplasmic,
title = {{Cytoplasmic SHMT2α promotes DNA damage repair and sustains cancer stemness to confer cisplatin resistance in non-small cell lung cancer: an in vitro study}},
author = {ZHANG Xinyu and GU Chen and LIU Yue and GUO Changcheng and ZENG Yuanyuan},
journal = {Zhongguo aizheng zazhi},
year = {2026},
month = sep,
url = {http://www.china-oncology.com/zh/article/doi/10.19401/j.cnki.1007-3639.2026.09.006/},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Cytoplasmic SHMT2α promotes DNA damage repair and sustains cancer stemness to confer cisplatin resistance in non-small cell lung cancer: an in vitro study
AU - ZHANG Xinyu
AU - GU Chen
AU - LIU Yue
AU - GUO Changcheng
AU - ZENG Yuanyuan
PY - 2026
DA - 2026/09/01
JO - Zhongguo aizheng zazhi
UR - http://www.china-oncology.com/zh/article/doi/10.19401/j.cnki.1007-3639.2026.09.006/
AB - MethodsCytoplasmic and mitochondrial proteins were extracted from A549 and A549-DDP cells, and the expression levels of SHMT2 in the respective subcellular fractions were detected by Western blot. Stable cell lines overexpressing cytoplasmic SHMT2α or knocking out SHMT2 were established. Cell viability following treatment with gradient concentrations of DDP (0.0, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0 μmol/L) for 48 h was assessed using the cell counting kit-8 (CCK-8) assay. Western blot and immunofluorescence assays were employed to evaluate the alterations in the protein expression of γ-H2AX, a pivotal biomarker for DNA damage repair, in stably transfected cells after treatment with 5 μmol/L DDP for 48 h. The expression of cancer stem cell markers was examined by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and a tumor stem cell spheroid formation assay was used to evaluate stemness maintenance capacity. Data were analyzed using GraphPad Prism (v8.0), and all experiments were independently repeated three times.ResultsThe total SHMT2 protein expression levels were similar between A549 and A549-DDP cells (P=0.129 6); however, compared to A549 cells, SHMT2 expression was higher in the cytoplasm of DDP-resistant cells (P=0.033 7). Overexpression of cytoplasmic SHMT2α led to an increased half-maximal inhibitory concentration (IC₅₀)（A549: P=0.007 7; H1299: P<0.001） and decreased γ-H2AX expression (A549: P=0.013 6; H1299: P=0.024 2) following 5 μmol/L DDP for 48 h, whereas SHMT2 knockout resulted in a decreased IC₅₀ (A549: sg-SHMT2-1: P=0.010 1; sg-SHMT2-2: P=0.006 6; H1299: sg-SHMT2-1: P=0.029 0; sg-SHMT2-2: P=0.013 7) and increased γ-H2AX expression (A549: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.015 5; H1299: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.002 4). Furthermore, overexpression of the cytoplasmic SHMT2α promoted the expression of cancer stem cell markers (A549: SOX2, CD133, BMI1: P<0.001; OCT4: P=0.006 8; Nanog: P=0.002 8; CD44: P=0.043 5; H1299: OCT4, Nanog, SOX2, CD133, CD44: P<0.001; BMI1: P=0.003 4) and enhanced the tumorsphere formation efficiency of NSCLC cancer stem cells (A549: P=0.012 8; H1299: P<0.001). Conversely, SHMT2 knockout reduced the levels of cancer stem cell markers (A549: SOX2, CD133: P<0.001; OCT4: sg-SHMT2-1: P=0.018 4; sg-SHMT2-2: P=0.048 5; Nanog: sg-SHMT2-1: P=0.002 9; sg-SHMT2-2: P=0.002 8; BMI1: sg-SHMT2-1: P=0.004 5; sg-SHMT2-2: P=0.003 5; CD44: sg-SHMT2-1: P=0.003 8; sg-SHMT2-2: P=0.002 7; H1299: OCT4, Nanog, SOX2, CD133, BMI1: P<0.001; CD44: sg-SHMT2-1: P=0.004 0; sg-SHMT2-2: P=0.004 3) and suppressed sphere formation efficiency (A549: P<0.001; H1299: P<0.001).ConclusionSHMT2 is highly expressed in the cytoplasm of DDP-resistant non-small cell lung cancer cells. It reduces cellular sensitivity to DDP by promoting DNA damage repair and sustaining tumor cell stemness, thereby providing a potential therapeutic target for reversing DDP resistance in non-small cell lung cancer.Background and purposeThe incidence and mortality of lung cancer have remained persistently high. Non-small cell lung cancer (NSCLC), the most common histological type, presents cisplatin (DDP) resistance as one of the most challenging clinical hurdles. SHMT2, a key metabolic enzyme that catalyzes the conversion of serine to glycine with concomitant generation of one-carbon units, is localized in both mitochondria and the cytoplasm and is highly expressed in various malignancies. This study aimed to investigate the role and molecular mechanism of cytoplasmic SHMT2α in DDP resistance of NSCLC.
SN - 1007-3639
ER -