Lichong Xiaozheng Granules enhances cisplatin sensitivity of ovarian cancer xenografts in rats by regulating adenine nucleotide translocator 3-mediated mitochondrial apoptosis

doi:10.12122/j.issn.1673-4254.2025.11.03

Abstract

Abstract:

Objective To investigate the molecular mechanism by which Lichong Xiaozheng Granules (LCXZ) sensitize ovarian cancer to cisplatin (DDP) treatment. Methods LC-MS analysis was used to identify the blood components of LCXZ after its administration in mice via gavage. In a BALB/c mouse model bearing subcutaneous ovarian cancer xenografts, the effects of daily gavage of distilled water (control group), intraperitoneal injection of DDP (5 mg/kg) once a week, or both DDP injection and daily LCXZK gavage (15 g/kg) on tumor growth were evaluated. Histopathological changes in the xenografts and kidneys were assessed with HE staining. RNA-seq was performed to identify the differentially expressed genes followed by KEGG pathway analysis. The changes in mitochondrial ultrastructure and expressions of mitochondrial apoptosis-related were examined with transmission electron microscopy and Western blotting. Results A total of 218 blood-borne components of LCXZ were detected by LC-MS. In the tumor-bearing mice, treatments with DDP and DDP combined with LCXZ redcued the tumor volume by 60.3% and 72.6% compared with that in the control group, respectively. Transcriptomic analysis revealed significantly upregulated ANT3 expression in both the two treatment groups. Molecular docking indicated that the main active components of LCXZ were capable of binding to adenine nucleotide translocator 3 (ANT3) with binding energies below -6 kcal/mol. Transmission electron microscopy showed obvious mitochondrial swelling and outer-membrane damage in the tumor cells in DDP-treated mice, and these changes were more pronounced in the combined treatment group. The expression levels of BAX, ANT3, cleaved caspase-3 and cleaved caspase-9 were increased, whereas BCL-2 expression was decreased significantly in the tumor cells in both the DDP and DDP+LCXZ groups. Conclusion LCXZ enhances the therapeutic efficacy of cisplatin against ovarian cancer xenografts in mice by promoting mitochondrial dysfunction and activating apoptotic signaling pathways via upregulating ANT3.

Key words: Lichong Xiaozheng Granules, adenine nucleotide translocator 3, mitochondrial apoptosis, chemo-sensitization, ovarian cancer

Yiliu CHEN, Min MA, Ran SU, Yinbin ZHU, Qing FENG, Jiali LUO, Weifeng FENG, Xianxin YAN. Lichong Xiaozheng Granules enhances cisplatin sensitivity of ovarian cancer xenografts in rats by regulating adenine nucleotide translocator 3-mediated mitochondrial apoptosis[J]. Journal of Southern Medical University, 2025, 45(11): 2309-2319.

Figures/Tables 9

Fig.1 HPLC-UV fingerprints and mass spectrometric analysis of Lichong Xiaozheng Granules (LCXZ). A, B: HPLC-UV fingerprint of LCXZ at 210 nm and 254 nm, respectively. C: High-resolution mass spectrometric fragmentation pathway of paeoniflorin.

Tab.1 Information on the top 20 compounds of Lichong Xiaozheng Granules (LCXZ) detected in medicated mouse serum

No.	Mass-to-charge ratio (m/z)	Formula	Mass Error (ppm)	Retention time (min)
M0001	387.1132	C12H22O11	-3.69	0.89
M0002	170.0210	C7H6O5	-3.02	1.87
M0003	329.0869	C14H18O9	-2.67	3.13
M0004	183.0293	C8H8O5	-3.03	3.90
M0005	356.1097	C16H20O9	-2.96	4.04
M0006	496.1569	C23H28O12	-2.36	4.15
M0007	457.1572	C20H27NO11	-2.76	4.29
M0008	431.1910	C19H30O8	-3.31	4.51
M0009	481.1692	C23H28O11	-2.65	4.62
M0010	295.1044	C14H17NO6	-4.10	4.67
M0011	480.1618	C23H28O11	-2.89	4.71
M0012	446.1200	C22H22O10	-2.88	4.88
M0013	428.1671	C20H28O10	-2.70	4.97
M0014	580.2141	C28H36O13	-2.51	5.12
M0015	433.1130	C21H22O10	-2.46	5.25
M0016	463.1221	C22H22O11	-3.07	5.26
M0017	441.1756	C20H28O8	-2.57	5.41
M0018	507.1491	C23H26O10	-3.62	5.58
M0019	161.0593	C10H10O3	-2.29	5.71
M0020	507.1493	C23H26O10	-3.22	5.90

Fig.2 LCXZ synergizes with cisplatin to inhibit ovarian cancer proliferation in nude mice. A: Measurement of tumor volume in the mice. B: Gross observation of the dissected tumors. C: Measurement of tumor weight. D: HE/Ki67 staining of the tumors,Arrows indicate widened intercellular spaces (Original magnification: ×400). *P<0.05 vs Tumor group, #P<0.05 vs DDP group (n=8).

Fig.3 LCXZ alleviates the toxic effect of cisplatin. A: Body weight changes of the tumor-bearing mice. B: Gross observation of the spleen of the mice. C: HE staining of renal tissues of the mice (×400). D: SOD content in the kidneys of the mice. *P<0.05, **P<0.01 vs Tumor group; #P<0.05 vs DDP group (n=8).

Fig.4 Transcriptomic analysis of therapeutic targets of LCXZ combined with cisplatin. A: Principal component analysis. B: Volcano plot of the differential genes of DDP vs Tumor. C: Volcano plot of the differential genes of DDP_LCXZ vs Tumor. D: Volcano plot of the differential genes of DDP_LCXZ vs DDP. E: Venn diagram of the differential genes.

Fig.5 KEGG enrichment analysis of the therapeutic targets of LCXZ combined with cisplatin. A: KEGG enrichment analysis of DDP vs Tumor. B: KEGG enrichment analysis of DDP_LCXZ vs Tumor. C: KEGG enrichment analysis of DDP_LCXZ vs DDP

Fig.6 Molecular docking of the main constituents of LCXZ. A: Paeoniflorin-ANT3 docking. B: L-amygdalin-ANT3 docking. C: Turanose-ANT3 docking. D: Benzoyl paeoniflorin-ANT3 docking.

Fig.7 LCXZ combined with cisplatin causes excessive opening of the mitochondrial pores in the tumor cells.( Left: scale bar=2 μm; Right: scale bar=1 μm. Labels: N:nucleus; M: mitochondria; ER:endoplasmic reticulum; ASS: autophagolysosomal structure).

Fig.8 LCXZ -mediated mitochondrial apoptosis pathway sensitizes cisplatin. A: Representitative protein bands in Westen blotting. B-F: Relative protein expression levels of ANT3, BAX, cleaved caspase-3, cleaved caspase-9 and BCL-2, respectively. *P<0.05, **P<0.01 vs Tumor group (n=3).

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