BACKGROUND: Identifying molecular biomarkers for predicting responses to anti-cancer drugs can enhance treatment precision and minimize side effects. This study investigated the novel cancer-targeting mechanism of combining SH003, an herbal medicine, with docetaxel in non-small cell lung cancer (NSCLC) cells. Also, the present study aimed to identify the genetic characteristics of cancer cells susceptible to this combination.
METHODS: Cell viability was analyzed by WST-8 assay. Apoptosis induction, BrdU incorporation, and cell cycle analysis were performed using flow cytometry. Metabolites were measured by LC-MS/MS analysis. Real-time PCR and western blotting evaluated RNA and protein expression. DNA damage was quantified through immunofluorescence. cBioPortal and GEPIA data were utilized to explore the mutual co-occurrence of TP53 and UMPS and UMPS gene expression in NSCLC.
RESULTS: The combination treatment suppressed de novo pyrimidine nucleotide biosynthesis by reducing the expression of related enzymes. This blockade of pyrimidine metabolism led to DNA damage and subsequent apoptosis, revealing a novel mechanism for inducing lung cancer cell death with this combination. However, some lung cancer cells exhibited distinct responses to the combination treatment that inhibited pyrimidine metabolism. The differences in sensitivity in lung cancer cells were determined by the TP53 gene status. TP53 wild-type lung cancer cells were effectively inhibited by the combination treatment through p53 activation, while TP53 mutant- or null-type cells exhibited lower sensitivity.
CONCLUSIONS: This study, for the first time, established a link between cancer cell genetic features and treatment response to simultaneous SH003 and docetaxel treatment. It highlights the significance of p53 as a predictive factor for susceptibility to this combination treatment. These findings also suggest that p53 status could serve as a crucial criterion in selecting appropriate therapeutic strategies for targeting pyrimidine metabolism in lung cancer.

OBJECTIVE: BRAF mutations are relatively uncommon in lung cancer. However, the majority of therapies targeting BRAF mutations have been developed exclusively for lung cancer patients with V600E mutations, limiting their effectiveness in treating tumors with the non-V600E BRAF mutations. As a result, there is a need to explore effective therapeutic strategies for patients with lung cancer carrying non-V600 BRAF mutations. Therefore, this study aims to identify a combination treatment approach that effectively targets lung cancer with G469A non-V600 BRAF alteration.
METHODS: The efficacy of drug treatments was assayed using a patient-derived xenograft (PDX) mouse model. Histological analysis was performed using hematoxylin and eosin and immunohistochemical staining. Cell viability and growth were determined using the WST-8 and colony formation assays. Protein levels and apoptosis were analyzed using western blot and flow cytometry, respectively.
RESULTS: We demonstrated that the lung cancer cells harboring the non-V600E G469A mutation were responsive to the combination of SH003 and dabrafenib. By utilizing patient-derived xenograft (PDX) models, we identified that this combined treatment induces apoptosis and exhibits antitumor effects through the reduction of ERK signals. The synergistic effect of the combination treatment on BRAF G469A lung cancer cells was consistent with its effects on PDX models, suggesting that the molecular mechanism of apoptosis involves a decrease in the MEK/ERK signaling pathway.
CONCLUSIONS: The SH003 and dabrafenib combination can be potentially developed as an effective treatment strategy for addressing lung cancer patients with the BRAF G469A mutation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of human death worldwide. Herbal prescription SH003 has been developed to treat several cancers including NSCLC. Due to the multi-component nature of SH003 with multiple targets and pathways, a network pharmacology study was conducted to analyze its active compounds, potential targets, and pathways for the treatment of NSCLC.
METHODS: We systematically identified oral active compounds within SH003, employing ADME criteria-based screening from TM-MC, OASIS, and TCMSP databases. Concurrently, SH003-related and NSCLC-associated targets were amalgamated from various databases. Overlapping targets were deemed anti-NSCLC entities of SH003. Protein-protein interaction networks were constructed using the STRING database, allowing the identification of pivotal proteins through node centrality measures. Empirical validation was pursued through LC-MS analysis of active compounds. Additionally, in vitro experiments, such as MTT cell viability assays and western blot analyses, were conducted to corroborate network pharmacology findings.
RESULTS: We discerned 20 oral active compounds within SH003 and identified 239 core targets shared between SH003 and NSCLC-related genes. Network analyses spotlighted 79 hub genes, including TP53, JUN, AKT1, STAT3, and MAPK3, crucial in NSCLC treatment. GO and KEGG analyses underscored SH003\'s multifaceted anti-NSCLC effects from a genetic perspective. Experimental validations verified SH003\'s impact on NSCLC cell viability and the downregulation of hub genes. LC-MS analysis confirmed the presence of four active compounds, namely hispidulin, luteolin, baicalein, and chrysoeriol, among the eight compounds with a median of > 10 degrees in the herb-compounds-targets network in SH003. Previously unidentified targets like CASP9, MAPK9, and MCL1 were unveiled, supported by existing NSCLC literature, enhancing the pivotal role of empirical validation in network pharmacology.
CONCLUSIONS: Our study pioneers the harmonization of theoretical predictions with practical validations. Empirical validation illuminates specific SH003 compounds within NSCLC, simultaneously uncovering novel targets for NSCLC treatment. This integrated strategy, accentuating empirical validation, establishes a paradigm for in-depth herbal medicine exploration. Furthermore, our network pharmacology study unveils fresh insights into SH003\'s multifaceted molecular mechanisms combating NSCLC. Through this approach, we delineate active compounds of SH003 and target pathways, reshaping our understanding of its therapeutic mechanisms in NSCLC treatment.

Melanoma is the most invasive and lethal skin cancer. Recently, PD-1/PD-L1 pathway modulation has been applied to cancer therapy due to its remarkable clinical efficacy. SH003, a mixture of natural products derived from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii, and formononetin (FMN), an active constituent of SH003, exhibit anti-cancer and anti-oxidant properties. However, few studies have reported on the anti-melanoma activities of SH003 and FMN. This work aimed to elucidate the anti-melanoma effects of SH003 and FMN through the PD-1/PD-L1 pathway, using B16F10 cells and CTLL-2 cells. Results showed that SH003 and FMN reduced melanin content and tyrosinase activity induced by α-MSH. Moreover, SH003 and FMN suppressed B16F10 growth and arrested cells at the G2/M phase. SH003 and FMN also led to cell apoptosis with increases in PARP and caspase-3 activation. The pro-apoptotic effects were further enhanced when combined with cisplatin. In addition, SH003 and FMN reversed the increased PD-L1 and STAT1 phosphorylation levels induced by cisplatin in the presence of IFN-γ. SH003 and FMN also enhanced the cytotoxicity of CTLL-2 cells against B16F10 cells. Therefore, the mixture of natural products SH003 demonstrates therapeutic potential in cancer treatment by exerting anti-melanoma effects through the PD-1/PD-L1 pathway.

OBJECTIVE: Breast cancer is one of the most common cancers in women all over the world and new treatment options are urgent. ER stress in cancer cells results in apoptotic cell death, and it is being proposed as a new therapeutic target. SH003, a newly developed herbal medicine, has been reported to have anti-cancer effects. However, its molecular mechanism is not yet clearly defined.
METHODS: Microarray was performed to check the differential gene expression patterns in various breast cancer cell lines. Cell viability was measured by MTT assays to detect cytotoxic effects. Annexin V-FITC and 7AAD staining, TUNEL assay and DCF-DA staining were analyzed by flow cytometry to evaluate apoptosis and ROS levels, respectively. Protein expression was examined in SH003-breast cancer cells using immunoblotting assays. The expression of C/EBP Homologous Protein (CHOP) mRNA was measured by real-time PCR. The effects of CHOP by SH003 treatment were investigated using transfection method.
RESULTS: Herein, we investigated the molecular mechanisms through which SH003 causes apoptosis of human breast cancer cells. Both cell viability and apoptosis assays confirmed the SH003-induced apoptosis of breast cancer cells. Meanwhile, SH003 altered the expression patterns of several genes in a variety of breast cancer cell lines. More specifically, it upregulated gene sets including the response to unfolded proteins, independently of the breast cancer cell subtype. In addition, SH003-induced apoptosis was due to an increase in ROS production and an activation of the ER stress-signaling pathway. Moreover, CHOP gene silencing blocked SH003-induced apoptosis.
CONCLUSIONS: SH003 causes apoptosis of breast cancer cells by upregulating ROS production and activating the ER stress-mediated pathway. Thus, our findings suggest that SH003 can be a potential therapeutic agent for breast cancer.

Exosomes released from tumor cells treated with cancer-targeting drugs reflect altered metabolic processes within the cells. Therefore, metabolites in exosomes can be used as markers to predict the therapeutic response or identify therapeutic targets. In this study, metabolite changes in exosomes were investigated by co-administration of the herbal extract SH003 and docetaxel (DTX), which exert a synergistic anti-cancer effect on lung cancer cells. Exosomes released from cells treated with SH003 and DTX were purified, and untargeted metabolic profiling was performed by liquid chromatography-tandem mass spectrometry. Analysis of altered metabolic-based pathways showed that the combined treatment synergistically increased pyrimidine metabolism compared with single-drug treatment. Additionally, xenobiotic metabolism by cytochrome P450 was specifically increased in cells treated with the combination. However, the released exosomes and increased metabolites in exosomes did not affect the anti-cancer effect of SH003 and DTX. Therefore, our study suggests that metabolite profiling can be used to evaluate the efficacy of combined treatments. Furthermore, such exosome-based metabolism may facilitate understanding the physiological endpoints of combination therapy in human biofluids.

BACKGROUND: A mixture (SH003) of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) has beneficial effects against several carcinomas. There have been few reports on an immune-enhancing activity of SH003 and its active constituent nodakenin.
OBJECTIVE: This study aimed at identifying the immune-enhancing effect of SH003 and nodakenin.
METHODS: The immune-enhancing effect was evaluated using RAW264.7 macrophages, mouse primary splenocytes, and a cyclophosphamide (CP)-induced immunosuppression murine model.
RESULTS: The results show that SH003 or nodakenin stimulated the production levels of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α, and nitric oxide (NO) and the expression levels of iNOS in RAW264.7 macrophages. SH003 or nodakenin also enhanced NF-κB p65 activation in RAW264.7 macrophages. SH003 or nodakenin stimulated the production levels of IFN-γ, IL-12, IL-2, TNF-α, and NO and the expression levels of iNOS in splenocytes. SH003 or nodakenin increased the splenic lymphocyte proliferation and splenic NK cell activity. In addition, SH003 or nodakenin increased the levels of IFN-γ, IL-12, IL-2, IL-6, and TNF-α in the serum and spleen of CP-treated mice, alleviating CP-induced immunosuppression.
CONCLUSIONS: Taken together, the results of this study show that SH003 improved immunosuppression through the activation of macrophages, splenocytes, and NK cells. These findings suggest that SH003 could be applied as a potential immunostimulatory agent for a variety of diseases caused or exacerbated by immunodeficiency.

Epidermal growth factor receptor (EGFR) is overexpressed in lung cancer patients. Despite treatment with various EGFR tyrosine kinase inhibitors, recurrence and metastasis of lung cancer are inevitable. Docetaxel (DTX) is an effective conventional drug that is used to treat various cancers. Several researchers have studied the use of traditional herbal medicine in combination with docetaxel, to improve lung cancer treatment. SH003, a novel herbal mixture, exerts anticancer effects in different cancer cell types. Here, we aimed to investigate the apoptotic and anticancer effects of SH003 in combination with DTX, in human non-small-cell lung cancer (NSCLC). SH003, with DTX, induced apoptotic cell death, with increased expression of cleaved caspases and cleaved poly (ADP-ribose) polymerase in NSCLC cells. Moreover, SH003 and DTX induced the apoptosis of H460 cells via the suppression of the EGFR and signal transducer and activator of transcription 3 (STAT3) signaling pathways. In H460 tumor xenograft models, the administration of SH003 or docetaxel alone diminished tumor growth, and their combination effectively killed cancer cells, with increased expression of apoptotic markers and decreased expression of p-EGFR and p-STAT3. Collectively, the combination of SH003 and DTX may be a novel anticancer strategy to overcome the challenges that are associated with conventional lung cancer therapy.

Background: Cancer is a major health problem worldwide and the leading cause of death in many countries. Preclinical studies have shown the therapeutic anticancer effects of SH003, a novel herbal medicine containing Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii. The present study investigated the maximum tolerated dose of SH003 in patients with solid cancers. Methods: This open-label, dose-escalation trial used the traditional 3 + 3 dose-escalation design. Patients with solid cancers were recruited and administered 1 to 4 tablets of SH003 thrice daily for 3 weeks according to the dose level. Adverse events were evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE). Dose-limiting toxicities (DLTs) were defined as Grade 3 or higher adverse events based on CTCAE. The maximum tolerated dose was defined as the highest dose at which no more than 1 of 6 patients experienced DLT. Results: The present study enrolled 11 patients. A total of 31 adverse events occurred. According to the CTCAE, all the observed adverse events were grade 2 or less and no adverse events of grade 3 or more corresponding to DLT occurred. Conclusion: The study results indicated that the maximum tolerated dose of SH003 was 4800 mg/day. A Phase 2 study is required to determine the efficacy of SH003 in patients with cancer at a dose of 4800 mg/day or less.

Drug markets revisits herbal medicines, as historical usages address their therapeutic efficacies with less adverse effects. Moreover, herbal medicines save both cost and time in development. SH003, a modified version of traditional herbal medicine extracted from Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes Kirilowii Maximowicz (Tk) with 1:1:1 ratio (w/w) has been revealed to inhibit tumor growth and metastasis on highly metastatic breast cancer cells, both in vivo and in vitro with no toxicity. Meanwhile, autophagy is imperative for maintenance cellular homeostasis, thereby playing critical roles in cancer progression. Inhibition of autophagy by pharmacological agents induces apoptotic cell death in cancer cells, resulting in cancer treatment. In this study, we demonstrate that SH003-induced autophagy via inhibiting STAT3 and mTOR results in an induction of lysosomal p62/SQSTM1 accumulation-mediated reactive oxygen species (ROS) generation and attenuates tumor growth. SH003 induced autophagosome and autolysosome formation by inhibiting activation of STAT3- and mTOR-mediated signaling pathways. However, SH003 blocked autophagy-mediated p62/SQSTM1 degradation through reducing of lysosomal proteases, Cathepsins, resulting in accumulation of p62/SQSTM1 in the lysosome. The accumulation of p62/SQSTM1 caused the increase of ROS, which resulted in the induction of apoptotic cell death. Therefore, we conclude that SH003 suppresses breast cancer growth by inducing autophagy. In addition, SH003-induced p62/SQSTM1 could function as an important mediator for ROS generation-dependent cell death suggesting that SH003 may be useful for treating breast cancer.