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Solamargine induces autophagy‐mediated apoptosis and enhances bortezomib activity in multiple myeloma

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy with a poor survival rate. Conventional chemotherapeutic agent‐induced adverse events, including toxicity, neuropathy or drug resistance, significantly decrease the patients' quality of life and can even lead to interruption of treatment. Therefore, novel therapeutic drugs and strategies are urgently needed to improve MM therapy and patient outcomes. Here, we show that solamargine (SM), a steroidal alkaloid glycoside isolated from a Chinese herb Solanum nigrum L., exhibits promising anti‐MM activity. In particular, SM suppressed the viability of MM cell lines (ARP‐1 and NCI‐H929) in a concentration‐ and time‐dependent manner, inducing apoptosis in these cells. RNA‐seq analysis showed that treatment with SM led to the upregulation of genes associated with cell death and autophagy in H929 cells. Further, we found that treatment with SM activated autophagy in the MM cells, as incubation with 3‐Methyladenine, an inhibitor of autophagy, significantly alleviated SM‐triggered apoptosis and inhibition of viability in MM cells. Interestingly, we also observed a synergistic effect between SM and bortezomib (BTZ), a common chemotherapeutic agent for MM, in both MM cells and human bone marrow CD138+ primary myeloma cells. We also confirmed the single‐agent efficacy of SM and the synergistic effects between SM and BTZ in an MM xenograft mouse model. Collectively, these findings indicate that SM exerts an anti‐MM effect, at least in part, by activating cell autophagy and reveal that SM alone or in combination with BTZ is a potential therapeutic strategy for treating MM.

leading to organ dysfunction. 1 Over 32,000 patients are newly diagnosed with MM in the United States every year, accounting for about 1% of all cancers and 10% of neoplastic haematologic tumours. 2,3 The survival of these patients has improved dramatically with the development of various novel treatment options, such as immuno-modulatory drugs, proteasome inhibitors, monoclonal antibodies and histone deacetylase inhibitors. 4,5 However, adverse events, including toxicity, neuropathy or relapse are still problems that severely restrict the treatment of MM. 6 Indeed, MM is still considered incurable, and almost 13,000 patients die of this disease each year in the United States. 3 Therefore, there is an urgent demand for new drugs and therapeutic strategies to improve the survival outcomes of MM patients.
Traditional Chinese medicine and its bioactive components play an important role in the treatment of many haematologic tumours.
Clinically, homoharringtonine, arsenic trioxide and etoposide have shown potent anti-cancer activity against acute myeloid leukaemia, acute promyelocytic leukaemia and Burkitt Lymphoma. [7][8][9] For the treatment of MM, some phytochemicals, including agaricus and curcumin, have also been found to exert significant anti-tumour effects by arresting the cell cycle, promoting anti-angiogenesis, or inducing apoptosis. 6 Thus, it is possible that components of natural plants, when extracted, could be used to treat MM and other haematologic tumours. Solamargine (SM, Figure 1A), a steroidal alkaloid glycoside isolated from the Chinese herb Solanum nigrum L., exhibits a promising anti-proliferative activity in various types of cancer cells, including those derived from human gastric cancer, lung cancer and nasopharyngeal carcinoma. [10][11][12] Nevertheless, the efficacy of SM in the treatment of MM has yet to be evaluated. In addition, while combination chemotherapy with multiple drugs is commonly used in the clinic to improve response rates, prolong response and delay the onset of drug resistance of tumour cells, the efficacy of SM in combination with other anti-MM agents has also not yet been examined.
Among the pathways by which many chemotherapy agents function, autophagy is an evolutionarily conserved intracellular degradation pathway in which protein aggregates and damaged organelles are removed via lysosomal degradation. 13 Autophagy is associated with cell survival/proliferation and is indeed beneficial for tumour maintenance, as it can recycle damaged organelles, overcome nutrient deprivation, relieve metabolic stress, and regulate cellular homeostasis. 14,15 However, excessive autophagy may result in cell death. 16 In MM, autophagy induced by chemotherapeutic drugs can either play a prosurvival and drug-resistant role by relieving cellular stress caused by toxic stimulation, or a subversive role by triggering autophagic cell death (ACD). 17 Thus, regulating autophagy to promote MM cell death F I G U R E 1 Chemical structure of solamargine (SM) (A) and traditional Chinese medicine symptom-Chinese medicine-target network of SM (B) F I G U R E 2 Legend on next page. and increase drug sensitivity may be a novel therapeutic strategy to combat this disease. 18 In the current study, we investigated the role of SM in the regulation of autophagy in MM cells and the efficacy of this natural agent at suppressing MM in vitro and in vivo. Furthermore, treatment with SM was combined with bortezomib (BTZ), a chemotherapeutic agent commonly used in the treatment of MM, to investigate this potential clinical application of SM in treating MM. We show for the first time that SM activates autophagy which contributes to apoptosis and suppression of viability in human MM cells (ARP-1 and NCI-H929). Moreover, the combination of SM and BTZ exhibits more effective tumour suppression than the treatment of SM or BTZ alone. Our findings thus identify SM, and particularly its combination with BTZ, as potential therapeutic options for the treatment of MM, and provide a theoretical basis for the potential clinical application of SM in MM therapy.

| SM suppresses cell viability and induces apoptosis in MM cells
It has been reported that SM suppresses the progression of lung cancer, gastric cancer and nasopharyngeal carcinoma, [10][11][12] whereas, to our knowledge, the effects of SM on MM has yet to be investigated.
We first examined for the potential of SM in treating MM using SymMap, which identifies overlap between known effects of traditional Chinese medicine components and a wide range of diseases. 19 Indeed, this analysis identified some overlap between SM and MM ( Figure 1B). Based on this, as well as the known efficacy of SM on other cancers, we investigated the effects of SM on MM cell lines. In particular, we examined the effects of SM at different concentrations on the viability of two MM cell lines, ARP-1 and H929 cells, after incubation for 24 h using the Cell Counting Kit-8 (CCK-8) assay. As shown in Figure  To further demonstrate the anti-MM effect of SM, cell apoptosis was measured by flow cytometry, following annexin-V-FITC/PI staining. As shown in Figure 2C Figure 3C). Interestingly, we also found that autophagy-related genes (red box) were dramatically upregulated in H929 cells treated with SM, suggesting that autophagy pathway may be involved in SM-induced suppression of MM.

| SM-induces apoptosis and suppression of viability in MM cells via autophagy
To determine whether indeed treatment with SM triggers autophagy in MM cells, we performed Western blot analysis, examining for the expressions of two autophagy-related proteins LC3-II and Beclin 1. We found that, consistent with the RNA-seq analysis, LC3-II and Beclin 1 were both significantly upregulated by SM in ARP-1 and H929 cells ( Figure 4A and B). We then examined the effects of a 2 h pre-treatment with 3-methyladenine (3-MA) (10 μM), an inhibitor of autophagy, followed by exposure to SM for 24 h in MM cells. As shown in Figure 4C  All quantified data were expressed as mean ± SD (n = 3-5, *p <0.05, **p <0.01) F I G U R E 3 Analysis of differential gene expression by RNA-seq data in solamargine (SM)treated H929 cells. (A) Consistency across replicates of the RNA-seq experiment. (B) The gene expression level was measured based on reads per kilobase of transcript per million mapped reads (RPKM). (C) Gene Ontology (GO) analysis of differentially expressed genes in H929 cells exposed to SM (the genes with at least two-fold change of expression level were identified) the detrimental effects of SM on MM cells is largely owing to an excessive activation of autophagy.   Figure 6A. Consistent with the in vitro observations, the combination of SM with BTZ caused a greater reduction in tumour growth than either single agent treatment alone ( Figure 6A). As shown in Figure 6B and C, after four consecutive treatments, the tumour size and weight were clearly decreased after treatment with SM or BTZ alone, compared with untreated control. Notably, the volumes and weights of the tumours were much lower in the group treated with BTZ plus SM ( Figure 6B and C). Moreover, we observed that the density of tumour cells and Ki-67 expression were strikingly decreased in tumour sections of SM-and BTZ-treated mice, as determined by HE and immunochemical staining, respectively ( Figure 6D). The combined treatment of SM and BTZ induced a further reduction in both the density of tumour cells and the expression of Ki-67 in tumour tissues ( Figure 6D). Of note, we observed a slight decrease in body weight in the mice treated with BTZ, whereas there was no significant difference in body weight between the SM treatment group and control, nor between the mice treated with the SM-BTZ combination and those treated with BTZ alone ( Figure 6E). Thus, taken together, these in vivo data further demonstrate the efficacy of SM in inhibiting the growth of MM as well as potent synergistic effects with BTZ, and indicate no significant toxicity caused by SM.

| DISCUSSION
There is presently a great need for novel therapeutic options in the treatment of MM owing to the poor survival rate of MM patients. 2,3 In this study, we explored the potential of SM, a component of the Natural products have been documented to exhibit many similarities to known metabolites as well as high levels of bioavailability. 6 Additionally, natural phytochemicals exert low, or even no, toxicity to healthy cells, and some have good pharmacokinetic properties. 20 Due to these factors, studies that report the potent effects of natural phytochemicals on cancers have increased dramatically in the last few decades. 6 Several studies have indicated that SM, a natural phytochemical component, is a promising anti-tumour drug in a variety of solid malignancies, including melanoma cancer, gastric cancer, pancreatic cancer, lung cancer and nasopharyngeal carcinoma. [10][11][12]21,22 In this work, we build on these observations and show that SM also Prior work has demonstrated a bidirectional role of autophagy in the pathophysiology of MM and in myeloma cells. 17,23 Emerging evidence has revealed that plasma cells heavily rely on autophagy during the differentiation of B cells. 23,24 A specific level of autophagy is necessary for myeloma cells to sustain life. 23,24 Hence, inhibiting autophagy is considered to be a potentially effective strategy against MM. 25 In most cases, autophagy plays an anti-apoptotic role by raising the stress threshold required for the induction of apoptosis, or by directly suppressing apoptosis. 17,26,27 However, in rare cases, excessive autophagy can lead to ACD of tumour cells due to the extreme degradation of the organelles and cytoplasm. 28,29 In the present study, using RNA-seq, we found that autophagy-related genes were transcriptionally activated by SM in H929 cells ( Figure 3C). Consistent with this, Western blotting analysis showed that SM markedly enhanced the levels of autophagy-associated proteins, LC3-II and Beclin 1, in both ARP-1 and H929 cells ( Figure 4A  and lucanthone, have been explored for cancer therapy. 30 Our study provides a contrary view whereby SM can be used as an autophagic activator in the treatment of MM. Of note, using SymMap, we predicted several potential targets of SM, including ABCG2, Bcl-2, PTEN, and RB1 ( Figure 1B). We then provided evidence that one of the predicted targets Bcl-2 was indeed downregulated by SM in MM cells ( Figure 2E and F). Bcl-2 plays a crucial role in autophagy inhibition by binding to Beclin 1 to impede autophagy induction. 25,31 Thus, it is possible that SM enhances autophagy by suppressing the level of Bcl-2 in MM cells. In solid tumours, non-coding RNAs including long non-coding RNAs and microRNAs play an important role in SMsuppressed tumour progression. [10][11][12] Numerous studies have revealed that non-coding RNAs participate in malignancy formation, progression, and metastasis in MM. [32][33][34] We thus speculate that non-coding RNAs may contribute to the excessive autophagy-mediated inhibition of MM cells exposed to SM, a possibility that remains to be further investigated.
Among first-line agents in the clinical treatment of MM, the proteasome inhibitor BTZ has significantly improved patient outcomes. 25 However, the current clinical issues with the use of BTZ are drug resistance and relapse. To improve response rates, prolong response and delay the onset of drug resistance, therapeutic combinations of BTZ with other agents have been frequently examined in preclinical studies and clinical MM therapy. 25 In this study, using the CCK-8 assay and CompuSyn software, we observed synergistic effects on MM between SM and BTZ ( Figure 5A). We also showed that the combination of SM plus BTZ was indeed more effective than either agent alone in MM cell lines (ARP-1 and H929) and human bone marrow CD138+ primary myeloma cells ( Figure 5B-D). These in vitro results were further confirmed in a MM xenograft mouse model (Figure 6), indicating that combining SM with BTZ increases tumour cytotoxicity and exhibits more significant anti-MM efficacy than either single agent alone. Interestingly, studies have validated that BTZ-elicited autophagy leads to drug resistance. 35,36 However, our study hinted that SM may sensitise MM cells to BTZ by further activating autophagy, suggesting that autophagy induction is a double-edged sword in the survival/growth/death of MM cells.
In summary, in this study, we have shown that SM, as a single agent, exhibits remarkable detrimental activity against MM at least partly by promoting autophagy, which provides a proof-of-principle for the potential clinical application of SM in the treatment of MM. We further demonstrated that the combination of SM with BTZ synergistically exerts a greater anti-MM effect in vitro as well as in vivo, with no significant toxicity of SM. Our results thus identify SM as a potential novel drug to treat MM with high efficiency and safety, and the combination of SM and BTZ as an additional promising therapeutic strategy against this disease. Finally, we note that, considering the marked capability of SM to combat a range of cancers shown in previous work, our present study further provides impetus to synthetic organic chemists to more deeply investigate the finer chemical details, and modification possibilities, of this natural alkaloid so as to produce even more potent therapeutic options against MM as well as other cancers.   The quality of raw sequence reads was examined by FastQC software. All qualified sequence reads were mapped to a reference genome (hg19) by TopHat. 38 Cufflinks was used to characterise the differential transcription pattern. 38 Gene expression level was measured by the reads per kilobase of transcript per million reads mapped (RPKM). For differential gene expression analysis, we identified the genes whose gene expression level saw at least a two-fold change.

| Chemicals and antibodies
The list of differentially expressed genes was uploaded to DAVID Bioinformatics Resources 6.7 and we performed analysis of the gene ontology (GO) cluster.

| Immunohistochemistry
Paraffin-embedded sections of tumour tissues from xenograft mice were incubated with an antibody directed against Ki-67 at 4 C overnight. Subsequently, the sections were treated with polymer, developed with DAB-Chromogen, and counterstained with haematoxylin, followed by imaging under a fluorescence microscopy (Leica DMi8, Wetzlar, Germany).

| Statistical analysis
All data were expressed as mean ± SD and calculated using Graphpad 8.0 (San Diego, CA, USA). One way analysis of variance followed by the Tukey's Post-hoc test were employed for statistical analysis. Differences with p-values less than 0.05 were considered statistically significant. Drug Synergy was quantified according to the Chou-Talalay Method for non-constant drug combination using the CalcuSyn software program (ComboSyn Inc., Paramus, NJ, USA). 39 A combination index (CI) >1.0 suggested antagonistic effect, a CI = 1 suggested additive effect, and a CI <1 suggested synergistic effect.