Combination of SH003 and paclitaxel modulates tumor microenvironment and inhibits metastasis of metastatic melanoma

Abstract

Developing therapeutic strategies to overcome immune evasion and resistance posed by the tumor microenvironment (TME) in advanced melanoma remains a significant challenge. This study evaluated therapeutic efficacy of an anticancer herbal extract SH003 combined with Paclitaxel (PTX) in a metastatic melanoma model. Results indicated that the combined therapy of SH003 and PTX not only bolstered antitumor immune responses by reducing influx of regulatory T cells (Tregs) and enhancing infiltration of cytotoxic T cells within the TME, but also significantly curtailed tumor growth and metastasis. Notably, the combined therapy of SH003 and PTX effectively inhibited the EGFR/JAK2/STAT3 and PI3K/AKT/mTOR signaling pathways, which are closely associated with tumor cell survival. Additionally, it reduced the expression of markers associated with metastasis, such as MMP-2, MMP-9, N-cadherin, CXCL9, and CXCL10. These findings suggest that SH003 in combination with PTX can reshape the TME, improve immune cell infiltration, and enhance antitumor immunity, offering a promising strategy to improve therapeutic outcomes of metastatic melanoma.

Keywords: Combination therapy; Melanoma; Metastasis; Paclitaxel; SH003; Tumor microenvironment.

Fig. 1

SH003 suppresses melanoma cell viability and migration by inducing apoptosis and modulating key regulators of metastasis. B16F10 cells were treated with SH003 at different concentrations (0, 100, 200, 400, 800, 1000 µg/mL) for 24 h. (A) Cell viability was measured by MTT assay. (B) The proportion of apoptotic cells was measured by flow cytometry with gating based on untreated controls after exclusion of debris and doublets. (C) Expression of melanin synthesis-related genes in B16F10 cells was analyzed at both protein and mRNA levels. Protein expression was assessed by Western blot (left) and quantified relative to GAPDH, whereas mRNA levels were measured by RT-qPCR (right), normalized to GAPDH, and presented as fold change relative to the control group. (D) Expression levels of apoptosis-associated proteins, including PARP, cleaved caspase-3, and Mcl-1, were analyzed by Western blot. B16F10 cells were treated with SH003 at indicated concentrations for 24 h. (E) CXCL9 and CXCL10 levels quantified with ELISA. (F) Expression levels of migration-associated proteins N-cadherin, MMP2, and MMP9 were assessed using Western blot. Statistical significance was determined using a one-way ANOVA (ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 2

SH003 and PTX synergistically suppress melanoma cell growth and induce apoptosis. (A) B16F10 melanoma cells were treated with SH003 (100 µg/mL) and PTX (10 nM) individually or in combination for 24 h and cell viability was assessed by MTT assay. (B) Synergistic effects of SH003 and PTX were quantified using SynergyFinder software. To detect apoptosis induced by SH003 and PTX, (C) the proportion of apoptotic cells was measured by flow cytometry with gating based on untreated controls after exclusion of debris and doublets. (D) Expression levels of apoptosis-associated proteins, including PARP, cleaved caspase-3, and Mcl-1, were analyzed by Western blot. Western blot analysis was used to determine expression levels of (E) EGFR/JAK2/STAT3 and (F) PI3K/AKT signaling pathway components. Statistical significance was determined using one-way or two-way ANOVA (ns, insignificant; * p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 3

Combination of SH003 and PTX suppresses metastatic potential of melanoma cells. B16F10 cells were treated with SH003 (100 µg/mL) and PTX (10 nM) individually or in combination for 24 h. (A, B) Levels of metastasis-related factors, including (A) CXCL9 and CXCL10, and (B) MMP-2, MMP-9, and N-cadherin, were quantified by ELISA and Western blot. Statistical significance was determined using a one-way ANOVA. (### p < 0.001 compared to the combination-treated group (SH003 + PTX); * p < 0.05, ** p < 0.01, *** p < 0.001, compared to non-treated group). (C, D) Migration and invasion abilities of B16F10 cells were assessed using (C) a migration assay, with cell intervals indicated by red lines and (D) an invasion assay. Images were taken at magnification x100. Statistical significance was determined using a two-way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 4

Combination of SH003 and PTX promotes intratumoral T-cell infiltration in melanoma. Therapeutic efficacy of combined SH003 and PTX treatment in 7-day-established B16 melanoma. B6 mice (6 per group) were inoculated subcutaneously (s.c.) with 1 × 10⁵ B16 cells on day 0. Treatments with SH003 (558 mg/kg, oral gavage) and PTX (5 mg/kg, intraperitoneally), either individually or in combination, were initiated on day 7. (A) Tumor sizes were determined for individual mice by measuring two opposing diameters. Results are presented as tumor areas in mm³. Statistical significance was determined using a two-way ANOVA. (*** p < 0.001, compared to the non-treated group). (B-D) In parallel with A, mice were sacrificed on day 21 to evaluate CD8⁺ T-cell responses and immune cell populations. Cells from disaggregated tissues of spleen and tumor were analyzed for compositions of various subsets of immune cells. (B) IFN-γ levels in spleens of individual mice were quantified with ELISA. (C) Tumors were dissociated into single-cell suspension to analyze immune and immunosuppressive cells. The proportion of Treg cells and CD8⁺ T cell/Treg ratio were calculated as the percentage of CD8⁺ T cells relative to CD4⁺Foxp3⁺ Tregs in each group. The left panel shows representative flow cytometry plots. (D) Tumor tissues were embedded in paraffin and stained for IFN-γ (yellow arrows) and DAPI to assess the localization and expression of IFN-γ within the tumor microenvironment by immunofluorescence. Immunofluorescence images were taken at magnification x400 (scale bar 20 μm and 5 μm; Carl Zeiss, Germany). Statistical significance was determined using a two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 5

Combination of SH003 and PTX suppresses lung metastasis. (A-C) To evaluate the in vivo efficacy of SH003 and PTX combination therapy in suppressing melanoma metastasis, B6 mice (5 per group) were inoculated intravenously (i.v.) with 5 × 10⁵ B16 cells on day 0. Starting on day 3, mice were treated with SH003 (558 mg/kg, oral gavage) and PTX (5 mg/kg, intraperitoneally), individually or in combination. On day 17, mice were sacrificed and (A) lung weights and pulmonary metastases were measured. The left panel shows a representative image of lung tissue containing pulmonary colonies from B16F10 melanoma cells. Lung tissues were stained with (B) hematoxylin and eosin and (C) immunohistochemistry for PCNA. Photo images were taken at magnifications x200 and x400 (scale bar 50 μm and 20 μm; Carl Zeiss, Germany). (D) Infiltrated CD8a in lung tissues were detected by immunohistochemistry. Photo images were taken at magnification x400 (scale bar 20 μm; Carl Zeiss, Germany). Quantification was performed using ImageJ by measuring mean intensity. Data are presented as fold change relative to control. (E) Lung tissues were embedded in paraffin and stained for IFN-γ (yellow arrows) and DAPI. Immunofluorescence images were acquired at 400 x magnification to assess localization and expression of IFN-γ (scale bar: 20 μm; Carl Zeiss, Germany). Quantification was performed using ImageJ by measuring integrated density. Data are presented as fold change relative to control. Statistical significance was determined using a two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 6

Evaluating effects of SH003 on immune responses in normal mice. Effects of SH003 on immune responses in normal mice were evaluated. B6 mice (10 per group) were treated with SH003 (558 mg/kg, oral gavage) for 14 days. (A) Body weight was measured twice weekly. (B) Spleen weight and index in mice were assessed at the end of the study. (C) Blood samples were collected via cardiac puncture and hematological analysis was performed using flow cytometry (Biotoxtech Co., Ltd., Ochang, Korea). (D) Immune cell composition in the spleen was analyzed by flow cytometry. (E) mRNA expression levels of toll-like receptors and (F) transcription factors in mouse spleen were measured by RT-PCR. Bars indicate the mean and standard deviation (SD) for each group of mice. Statistical significance was determined using Student’s t-test (ns, insignificant; *p < 0.05, **p < 0.01, ***p < 0.001)