Shengmai San improves osimertinib resistance of non-small cell lung cancer cells by regulating the lactate/Wnt/β-catenin/LDHA pathway

doi:10.12122/j.issn.1673-4254.2026.03.06

Abstract

Abstract:

Objective To explore the effect of Shengmai San (SMS) for improving osimertinib resistance in non-small cell lung cancer cells and the underlying mechanism. Methods Cultured A549 cells were treated with osimertinib alone or in combination with SMS-medicated rat serum, and the changes in cell viability and glucose and lactate levels were determined. The effect of SMS combined with osimertinib for improving osimertinib resistance of A549 cells was assessed in a mouse model bearing subcutaneous A549 cell xenograft. Western blotting, RT-qPCR, and immunofluorescence staining were used to analyze the effects of SMS and lactate on the Wnt/β‑catenin/LDHA signaling pathway. Results SMS significantly increased osimertinib sensitivity of A549 cells in a concentration-dependent manner. Compared with osimertinib alone, the combined treatment with SMS and osimertinib significantly inhibited cell viability, colony formation ability, and tumor growth in nude mice. SMS concentration-dependently decreased glucose and lactate levels in A549 cells. The results of Western blotting showed that SM inhibited the protein expression of LDHA, total β‑catenin, and cytoplasmic and nuclear β‑catenin, while lactate obviously activated the expressions of total β‑catenin protein, nuclear β‑catenin, and LDHA in A549 cells. Immunofluorescence concentration-dependent staining showed that lactic acid activated nuclear accumulation of β‑catenin protein, while SMS significantly inhibited the expression of nuclear β‑catenin protein; RT-qPCR demonstrated that lactate significantly increased mRNA expressions of the Wnt/β‑catenin downstream target genes (c-myc, CD44, Axin2, Oct3/4, survivin, and CCND1), while SMS inhibited their expressions. Conclusion SMS improves osimertinib resistance in non-small cell lung cancer cells by inhibiting lactate/Wnt/β-catenin/LDHA pathway-mediated glycolysis.

Key words: Shengmai San, non-small cell lung cancer, osimertinib resistance, glycolysis, lactate, mechanism Wnt/β?catenin pathway, lactate dehydrogenase A

Zhiqing LIANG, Fuzhen PAN, Liqiang DENG, Zhefen MAI, Yun MA, Chuanjian SHI, Weiming FU. Shengmai San improves osimertinib resistance of non-small cell lung cancer cells by regulating the lactate/Wnt/β-catenin/LDHA pathway[J]. Journal of Southern Medical University, 2026, 46(3): 523-531.

Figures/Tables 8

Tab.1 Primer sequence for RT-qPCR

Gene name		Sequence (5' to 3')
β-actin	Forward	GTCATTCCAAATATGAGATGCGT
	Reverse	GCTATCACCTCCCCTGTGTG
c-Myc	Forward	TTCGGGTAGTGGAAAACCAG
	Reverse	CAGCAGCTCGAATTTCTTCC
CD44	Forward	TCAGAGGAGTAGGAGAGAGGAAAC
	Reverse	GAAAAGTCAAAGTAACAATAACAGTGG
Axin-2	Forward	GGATCGCTCCTCTTGAAGGA
	Reverse	CTTTCGCCAACCGTGGTT
Survivin	Forward	CCACCGCATCTCTACATTCAAG
	Reverse	CAAGTCTGGCTCGTTCTCAGTG
Oct3/4	Forward	TCGAGAACCGAGTGAGAGGC
	Reverse	CACACTCGGACCACATCCTTC
Cyclin D1	Forward	CTGGAGGTCTGCGAGGAACA
Cyclin D1	Reverse	CCTTCATCTTAGAGGCCACGAA
LDHA	Forward	ATGGCAACTCTAAAGGATCAGC
LDHA	Reverse	CCAACCCCAACAACTGTAATCT

Fig.1 Viability and colony-forming ability of A549 cells with different treatments (Mean±SD, n=3). A: Viability of A549 cells treated with SMS, osimertinib, or osimertinib combined with various concentrations of SMS. B: Colony formation of A549 cells and quantitative analysis. ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs Osi group. SMS-L: SMS low-dose group; SMS-M: SMS medium-dose group; SMS-H: SMS high-dose group; Osi: Osimertinib group.

Fig.2 Assessment of xenograft growth in nude mice (Mean±SD, n=3). A-C: Representative images of xenograft tumors, growth curve and tumor weight in the tumor-bearing C57BL/6 mice in the 4 treatment groups. D: Changes in body weight of the mice in the 4 treatment groups. E: Immunohistochemistry for Ki67 in the tumor tissues in the 4 groups. *P<0.05, **P<0.01, ***P<0.001 vs Control group; #P<0.05, ###P<0.001 vs Osi group. a: Control group; b: SMS Medium-dose group; c: Osimertinib monotherapy group; d: SMS medium-dose combined with osimertinib group.

Fig.3 Glucose (A) and lactate (B) concentrations A549 cells in each group (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs Control group; ##P<0.01, ###P<0.001 vs Osi group. a: Control group; b: SMS Medium-dose group; c: Osimertinib monotherapy group; d: SMS medium-dose combined with osimertinib group.

Fig.4 Protein and mRNA expressions of the Wnt/β-catenin/LDHA signaling pathway in A549 cells in different groups (Mean±SD, n=3). A, B: Western blotting of β-catenin and LDHA expression levels in A549 cells. C: Western blotting of β-catenin expression and quantitative analysis its cytoplasmic and nuclear fractions. D: mRNA expressions of Wnt/β-catenin pathway genes detected by by RT-qPCR. *P<0.05, **P<0.01,***P<0.001 vs Control group. a: Control group; b: SMS Medium-dose group; c: Osimertinib monotherapy group; d: SMS medium-dose combined with osimertinib group.

Fig.5 Immunohistochemistry of β-catenin protein expression in the tumor tissues in different groups (Mean±SD, n=3). **P<0.01, ***P<0.001 vs Control group;###P<0.001 vs Osimertinib group. a: Control group; b: SMS Medium-dose group; c: Osimertinib monotherapy group; d: SMS medium-dose combined with osimertinib group.

Fig.6 Expression of Wnt/β-catenin/LDHA pathway components in A549 cells with different treatments (Mean±SD, n=3). A: Western blotting of β‑catenin and LDHA expression in control and 10 mmol/L lactate-treated cells. B: Western blotting of cytoplasmic and nuclear fractions of β‑catenin in control and 10 mmol/L lactate-treated cells. C: mRNA expression of Wnt/β-catenin downstream targets detected by RT-qPCR. *P<0.05, **P<0.01, ***P<0.001 vs Control group. a: Control group; e: 10 mmol/L lactate treatment group.

Fig.7 Immunofluorescence staining for detecting β-catenin expression in A549 cells. A: Control group; B: SMS-Medium-dose group; C: Osimertinib monotherapy group; D: SMS medium-dose combined with osimertinib group; E: 10 mmol/L lactate treatment group.

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