Molecular entities for the anti-melanoma effect of Solanum nigrum

TL;DR

This paper identifies specific compounds in Solanum nigrum that may help fight melanoma by targeting certain genes.

Contribution

The study discovers two unique active compounds in Solanum nigrum and their associated gene targets for melanoma.

Findings

Solanum nigrum has two active ingredients, diosgenin and solanocapsine, with anti-melanoma properties.

Three gene targets (CYP3A4, GBA2, and PTK6) are associated with the anti-melanoma effect of these compounds.

Abstract

Prognosis for advanced and metastatic melanoma remains poor despite low incidence rate and early diagnosis. A significant number of
melanoma patients use complementary and alternative medicines during their normal treatments in anticipating improving therapeutic
efficacy. Solanum nigrum shows effective inhibitory activity against melanoma cells compared to Hedyotis
diffusa, Scutellaria barbata, and Lobelia chinensis. Therefore, it is of interest to explore
the anti-melanoma entities of S. nigrum. Our data show that three unique melanoma-related gene targets (CYP3A4, GBA2
and PTK6) for two unique active ingredients (diosgenin and solanocapsine) present in S. nigrum have potential role.

Full text

Background:

Melanoma, which initiates from carcinogenesis of melanocytes, is the most serious type of skin cancer. Globally, 325,000 new melanoma cases and 57,000 melanoma-related deaths were estimated to occur in 2022 [1]. Although melanoma has a low incidence rate and is usually diagnosed in early stages, prognosis for metastatic melanoma is extremely poor [2, 3]. Currently, melanoma limited in the skin is usually removed by surgery. Melanoma spread beyond the skin is mainly treated with chemotherapy, radiotherapy, immunotherapy, and targeted therapy in addition to surgery [3]. To achieve better therapeutic efficacy, combination therapies, such as poly-chemotherapy and poly-immunotherapy, are also commonly administered in melanoma treatment [4]. Chemotherapy agents include dacarbazine, temozolomide, carboplatin, paclitaxel, cisplatin and vinblastine, whereas immunotherapy agents include immune checkpoint inhibitors such as ipilimumab, nivolumab and pembrolizumab and cytokines such as interferon α-2b and interleukin-2. However, these therapies are associated with high cost, severe toxicity, undesirable side effects, and rapid development of drug resistance (especially for metastatic melanoma) [5, 6, 7]. Thus, many melanoma patients, specifically patients from low-income countries, seek complementary and alternative medicine (CAM) treatments. A multicenter cross-sectional study of 1089 melanoma patients in Germany reported that about 40% of the patients showed an interest of using CAM and nearly 20% of the patients used CAM [8]. Natural health products and medicinal plant extracts have been widely used to treat different types of illnesses and diseases throughout the world. Various phytochemicals have also been shown to possess potent anticancer effect from both preclinical and clinical studies [9, 10-11]. Medicinal plants have emerged to be a valuable resource for identifying and developing anticancer therapeutics. In fact, 146 out of 174 anticancer drugs in the market between 1981 and 2014 have biological or natural product origins [12]. However, CAM usage (herbs, plant extracts, etc.) in cancer treatment faces several limitations. The major limitation is lack of understanding on the underlying molecular mechanisms of these products. The second limitation is insufficient molecular profiling of the plant components. Finally, information on molecular interactions among different components in CAM products and between CAM products and drugs, foods and regulator molecules in human body is very limited. Hence, it is of interest to explore CAM products, especially their respective mechanism of action (MOA), which will not only improve therapeutic efficacy of the treatments but also protect patients' safety. We evaluated the anticancer activities of Hedyotis diffusa, Scutellaria barbata, Lobelia chinensis and Solanum nigrum against human melanoma cell line A-375 in our previous study [13]. Surprisingly, only the water extract of S. nigrum (WESN) possessed potent activity and exhibited a synergistic effect with chemotherapy drug temozolomide, whereas the most-commonly used anticancer herbs, H. diffusa and S. barbata, elicited little activity. Therefore, it is of interest to explore the molecular entities for the anti-melanoma effect of S. nigrum.

Methodology:

Active ingredients and corresponding targets in human cells:

A methodology flowchart of the current study was presented in supplementary Figure 6 (see PDF). Information of bioactive compounds for each medicinal plant (Table 4) was retrieved from the TCMSP database and analysis platform (https://tcmspw.com/tcmsp.php) using Hedyotis diffusa, Scutellaria barbata, Lobelia chinensis or Solanum nigrum as a keyword. Oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) were set as the thresholds in identifying candidate active ingredients [14, 15]. For each candidate, we obtained the structural graphic (sdf format) from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Then, the sdf file was uploaded to the Swiss Target Prediction database (http://swisstargetprediction.ch/index.php) to identify potential interacting protein targets for the candidate compound [16]. Target protein names filtered with "Homo sapiens" and "Probability > 0" were downloaded. The protein targets for the active ingredients in each medicinal herb were summarized in supplementary Table 5.

Melanoma-related targets:

Melanoma-related gene targets were retrieved from the GeneCards database (https://www.genecards.org/) using "melanoma" as a searching keyword. These targets were subsequently standardized in UniProt (https://www.uniprot.org/) with organism selected as "Homo sapiens" to obtain a list of human melanoma-related gene targets.

Unique active ingredients:

Unique active ingredient refers to a bioactive compound that is present in one medicinal plant but not in the others. From supplementary Table 4, we identified 4, 23, 15 and 4 unique active ingredients in H. diffusa, S. barbata, L. chinensis and S. nigrum, respectively (Table 1).

Unique targets for unique active ingredients:

For each medicinal plant, we first extracted the molecular targets for the unique active ingredients from supplementary Table 5. Then, unique molecular targets modulated by the unique active ingredients (Table 6) were obtained by removing shared molecular targets between/among medicinal plants. Finally, we identified unique melanoma-related targets for the unique active ingredients of each medicinal herb (Table 2) by extracting the common targets shared between the targets in supplementary Table 6 and human melanoma-related targets. We further carried out a reverse mapping of the 10 melanoma-related targets unique for S. nigrum for the corresponding unique active ingredients (Table 3).

Gene expression profiling in melanoma:

From the above analyses, we identified 10 unique melanoma-related gene targets that were modulated by two unique active compounds, diosgenin and solanocapsine, in S. nigrum. We then analyzed the expression of these 10 genes in melanoma patients (TCGA-SKCM dataset) using online software GEPIA (http://gepia.cancer-pku.cn/) that can analyze RNA sequencing expression data from both TCGA and GTEx projects [17]. Three genes (CYP3A4, GBA2 and PTK6) were identified to be significantly downregulated in melanoma patients. We further calculated survival curves (Kaplan-Meier plots) for these three genes in melanoma patients (TCGA-SKCM). Only gene PTK6 exhibited survival benefits at low level of expression.

Results:

H. diffusa, S. barbata, L. chinensis and S. nigrum are widely used to treat different types of cancer in traditional Chinese medicine (TCM). Particularly, H. diffusa and S. barbata are often used in pair, such as "kang ai ping wan" [18]. However, information on the anti-melanoma effect of these four medicinal herbs is very limited. Wang et al. reported that S. nigrum water extract could inhibit metastasis of melanoma cells in mice [19] and Chen et al. reported that the total flavonoids of S. barbata could inhibit melanoma growth by inducing autophagy and apoptosis [20]. However, Chen et al. used extracted flavonoids and it is unclear whether the content of S. barbata flavonoids in normal TCM formulas could reach to the level used in their experiment. Thus, we decided to undertake a comparative study of the four anticancer herbs against human melanoma A-375 cells and showed that only the water extract of S. nigrum (WESN) exhibited potent activity [13]. We further showed that WESN reduced intracellular ROS (reactive oxygen species) generation, which could lead to a cytostatic effect on the A-375 cells. Hence, in the current study, we embark on identifying unique melanoma targets for each herb and mapping them onto its respective active ingredients using a system pharmacology approach (Figure 6 - see PDF).

Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform is a database on systems pharmacology for herbal medicine, consisting of a collection of 499 Chinese herbs along with their respective bioactive ingredients, potential molecular targets and associated diseases [21]. Using this platform, we identified 7 active ingredients in H. diffusa, 29 active ingredients in S. barbata, 17 active ingredients in L. chinensis and 7 active ingredients in S. nigrum (Figure 1 - (see PDF) and Table 4). Only one compound, MOL000098 (quercetin), is shared among the four medicinal herbs. Unique active ingredients for each individual herb were summarized in Table 1. We identified 4 unique ingredients in H. diffusa, 23 in S. barbata, 15 in L. chinensis and 4 in S. nigrum (Table 1). Then, we extracted the molecular targets regulated by the active compounds of the four herbs (Figure 2 - (see PDF) and Table 5).

H. diffusa, S. barbata, L. chinensis and S. nigrum modulate 299, 299, 385 and 199 targets, respectively, out of which 146 targets are shared across the four herbs. We further identified that unique ingredients in H. diffusa modulate 82 unique targets, unique ingredients in S. barbata modulate 51 unique targets, unique ingredients in L. chinensis modulate 116 unique targets, and unique ingredients in S. nigrum modulate 27 unique targets, respectively (Table 6). To figure out why only S. nigrum is active against melanoma cells, we extracted 8144 melanoma-related gene targets from GeneCards and chose 4352 genes for further analysis after one cycle of median operation (score ≥ 0.69). Upon cross matching with targets modulated by the herbs, we identified 170 median melanoma-related targets from H. diffusa, 178 targets from S. barbata, 210 targets from L. chinensis and 112 targets from S. nigrum, respectively, out of which 87 gene targets are shared by all four herbs (Figure 3 - see PDF). Our further analysis showed that there are 41 gene targets unique for H. diffusa, 34 for S. barbata, 55 for L. chinensis and 10 for S. nigrum (Table 2).

Reverse mapping the 10 melanoma-related gene targets from S. nigrum to the active ingredients reveals that these 10 targets are modulated by 2 unique active compounds, diosgenin and solanocapsine (Table 1). This implicates that these two unique components from S. nigrum are likely to be responsible for the anticancer effect of WESN against the melanoma A-375 cells. Moreover, we performed a gene expression profiling for the 10 genes using TCGA-SKCM (skin cutaneous melanoma) dataset. Using | log_2_FC | ≥ 1 and p < 0.05 as the cutoff, three gene targets, CYP3A4, GBA2 and PTK6, were downregulated in skin cutaneous melanoma patients (Figure 4 - see PDF).

Discussion:

As shown in Table 3, target genes CYP3A4 and PTK6 are modulated by diosgenin. CYP3A encodes cytochrome P450 3A4 (CYP3A4), one of the most important liver drug-metabolizing enzymes. CYP3A4 is also expressed in skin cells and involved in skin metabolism of drugs [22]. Our current analysis is consistent with a previous study by Sumantran et al. that gene CYP3A4 was downregulated in melanoma [23]. In addition, Manda et al. reported that diosgenin inhibited CYP3A4 at IC50 of 17 mM [24]. Thus, we may hypothesize that S. nigrum could further inhibit the already downregulated CYP3A4 in melanoma cells, which, in turn, would reduce skin metabolism of drugs including chemotherapy agents and be beneficial for melanoma treatments. However, S. nigrum contains many compounds and the function of diosgenin can be interfered by the other compounds. Further studies are needed to identify how S. nigrum modulates the expression and function (upregulation or downregulation) of the three identified genes in its TCM formulations. PTK6 encodes the non-receptor tyrosine kinase 6 (PTK6 or BRK). PTK6 is overexpressed in breast cancer and deemed as an oncogene to promote cancer cell growth, survival, and migration [25- 26]. However, its expression is downregulated in melanoma patients. Recently, Fei and Chen proposed that PTK6, CAPNS1, DAPK2 and PARP1 formed an autophagy-related risk model, which could be used to evaluate prognosis of melanoma patients [27]. Furthermore, Kaplan Meier survival plot for patients included in the TCGA-SKCM dataset shows that high expression of PTK6 is associated with poor prognosis (Figure 5 - see PDF). Hence, we suggest that the downregulation of PTK6 in SKCM patients is potentially a response for maintaining homeostasis as autophagy has been reported to play an important role in maintaining skin homeostasis at cellular level [28, 29, 30-31]. Other than CYP3A4 and PTK6, other genes such as AKT1 and CDK1 have been identified as targets for diosgenin from both our current analysis and previous reports [32, 33]. However, these targets are unlikely to be responsible for the anti-melanoma activity of S. nigrum as they are also regulated by other medicinal plants such as H. diffusa, S. barbata and L. chinensis. Therefore, we conclude that genes CYP3A4 and PTK6, which are modulated by diosgenin, are likely to be the major targets conferring S. nigrum the anti-melanoma effect.

The third downregulated gene target GBA2 encodes non-lysosomyl glucosylceramidase β2 (GBA2), which catalyzes the hydrolysis of glucosylceramide into ceramide and glucose. It is modulated by solanocapsine, which is another unique active ingredient in S. nigrum. It has been shown that inducing GBA2 expression would promote glucosylceramide hydrolysis and impair melanoma growth in a mouse xenograft model [34]. Ceramide is an essential component of the highly evolutionarily conserved sphingolipid metabolism pathway. It forms a rheostat with sphingosine-1-phosphate (S1P), another key and potent sphingolipid metabolite [35]. The ceramide-S1P rheostat regulate a broad range of biological functions, such as ROS generation, cell proliferation and survival, cell apoptosis and cell autophagy [36, 37-38]. A shift of the rheostat towards ceramide side induces cell apoptosis and autophagy. Thus, GBA2 is highly like to exert its functions on cancer cell growth via regulating ceramide level in the ceramide-S1P rheostat. However, the modulation mechanism by solanocapsine on either GBA2 or cancer cell growth is still unknown. Further studies are warranted to identify the biological pathways regulated solanocapsine in human body.

Conclusion:

The anti-melanoma effect of S. nigrum is likely through the modulation of three unique melanoma-related gene targets (CYP3A4, GBA2 and PTK6) by its two unique active ingredients (diosgenin and solanocapsine). However, it should be noted that there are two major limitations: 1) the anti-melanoma effect of S. nigrum is possibly via the coordination of multiple active ingredients and may not be accurately represented by modulations from a single active ingredient and 2) crosstalk between different signaling pathways was not studied in the current research. Therefore, future transcriptomic and/or proteomic studies may provide a more accurate mechanism of action (MOA) for S. nigrum and illustrate the different types of crosstalk among signaling pathways regulated by S. nigrum.

Competing interests:

The authors declared no competing interests with respect to the research, authorship, and/or publication of this article.

Funding:

The authors disclosed no financial support for the research, authorship, and/or publication of this article.

Ethical approval:

Not applicable.

Author contributions:

Research idea: J Yang; Extraction of information on herbal active ingredients: Y Song; Analysis of melanoma targets: N Sakharkar; Data analysis: N Sakharkar, R Kaul-Ghanekar, Y Song, J Yang; Manuscript writing: N Sakharkar, J Yang.