Apoptosis is a type of programmed cell death, which can maintain normal cellular functioning and embryonic development by promoting cell death induced by multiple stimuli. Apoptosis can be induced by death receptor-dependent exogenous and mitochondria-dependent endogenous apoptotic pathways, and the process principally consists of alterations in mitochondrial outer membrane permeability, and the activation of a series of cysteinyl aspartate specific proteinase (caspase) and catabolic hydrolase (42). A prospective cohort study indicated that the apoptosis index, Ki-67 index and the ratio between the two, could serve as auxiliary assessments for the efficacy and prognosis of chemotherapy in patients with gastric cancer undergoing perioperative chemotherapy and radical gastrectomy (43). Furthermore, the avoidance of apoptosis is one of the hallmarks of chemotherapy resistance (44). The key to the occurrence of endogenous apoptosis lies in the balance between pro-apoptotic and pro-survival protein regulators (e.g., Bax and Bcl-2) (45). Notably, the upregulation of pro-survival proteins, such as Bcl-extra large and myeloid cell leukemia 1, has been suggested as one of the main reasons for the survival and drug resistance of various tumor cells (46). In addition, Bcl-2 can disrupt apoptotic signaling and ultimately inhibit the activation of caspases and apoptosis by preventing the release of cytochrome c from mitochondria (47).

Autophagy is a highly conserved intracellular catabolic process that occurs to degrade and eliminate misfolded proteins and damaged organelles, which is essential for maintaining metabolic homeostasis and energy balance. The process of autophagy mainly includes three stages: Phagocytic bubble assembly, autophagy formation and autophagy lysosome degradation (48). Depending on how it happens, autophagy has been mainly classified into three modes: Macroautophagy, microautophagy and chaperone-mediated autophagy (49). Autophagic cell death serves an important role in the action of antineoplastic drug therapy. However, during tumorigenesis and progression, autophagy has dual effects: In the early stage of tumor development, excessive autophagy induces autophagic cell death; by contrast, in cells in the middle and late stages, an increased level of autophagy promotes tumor survival and malignancy (50). Mammalian target of rapamycin (mTOR) kinase is an essential regulator of autophagy, which can be activated by the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)/mTOR pathway to inhibit autophagy, and can also promote autophagy through the negative regulation of the AMPK/mTOR pathway (51). Several studies have shown that the inhibition of autophagy can significantly enhance the sensitivity of tumor cells to chemotherapeutic agents and reverse drug resistance (52–54).

EMT is a process of change in which tumor cells lose epithelial characteristics and acquire a mesenchymal phenotype (55). The intrinsic mechanism is primarily associated with the specific loss of the epithelial marker E-cadherin and cell polarity, and the acquisition of the mesenchymal marker N-cadherin by tumor cells, including the activation of transcription factors such as Twist and Snail, and the expression of vimentin proteins (56). It has been shown that the occurrence of EMT might be related to a variety of tumor events, including tumorigenesis, deterioration, migration, invasion, acquisition of tumor stemness and drug resistance (57–59). EMT is a key step in inducing the formation of cancer stem cells (CSCs). The signaling pathways that activate EMT exhibit similarities with those that drive CSCs, such as the Wnt, Hedgehog and Notch pathways. Once EMT occurs, tumor cells will exhibit characteristics similar to those of CSCs, such as increased efflux of intracellular drugs and enhanced anti-apoptotic effects (60,61), thereby facilitating the survival and drug resistance of tumor cells.

The phenotype of drug resistance in tumor cells is usually associated with the upregulation of members of the ABC transporter protein family, especially P-glycoprotein (P-gp) encoded by the ABCB1 gene (62), MDR-related protein 1 (MRP1) encoded by the ABCC1 gene and breast cancer resistance protein (BCRP) encoded by the ABCG2 gene (63). These transporter proteins act as drug efflux pumps to catalyze the efflux of chemotherapeutic agents, thus contributing to the decreased levels of intracellular drug concentrations, and therefore attenuating the antitumor effects of drug therapy. It has been reported that the upregulation of P-gp, MRP1 and BCRP may give rise to poor clinical response and drug resistance in a variety of cancer types, such as human ovarian cancer, colon cancer, NSCLC and pancreatic cancer (64,65).

The mechanisms of drug resistance arising in tumor cells are complex, and there may be multiple mechanisms that intersect on one pathway to jointly mediate drug resistance in tumors. For example, the PI3K/AKT/mTOR signaling pathway, also referred to as the PAM axis, which is one of the most vital pathways regulating the basic physiological functions of cells, has a complex cascade that has an important role in the regulation of cell growth, differentiation, apoptosis, proliferation and metastasis (66). In general physiological and pathological processes, the PI3K/AKT/mTOR pathway works by transmitting signals from the upstream regulatory proteins, such as phosphatase and tensin homologue, PI3K and receptor tyrosine kinases, to a number of downstream effectors, such as mTOR, glycogen synthase kinase-3β, forkhead box O and mouse double minute 2 proteins (67). However, the hyperactivation and alteration of this pathway are often associated with the survival, proliferation, invasion and migration of tumor cells, further influencing the outcome of targeted therapy in human cancer (68). Due to PI3K being a primary drug target for cancer therapy, the development of more optimized PI3K inhibitors has consistently been a direction in the field of anticancer drug development. However, the rapidly accelerated fibrosarcoma (RAF)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is another pathway that exerts a similar function to the PI3K/AKT/mTOR pathway (69), and a cross-inhibitory pattern exists between these two pathways, which exerts negative regulation on each other's activity. Therefore, when one pathway is chemically blocked, it releases the cross-inhibition and effectively activates the other pathway to synergistically mediate cell survival (Fig. 3) (67,70). Consequently, the complex crosstalk between signaling pathways is strongly associated with the refractoriness of tumors; however, a study has shown that the development of drugs targeting these crosstalk-pathway regulatory factors offers a new strategy for solving this problem (71).

Timosaponin AIII (TS-AIII) (Fig. 2A) is a steroidal saponin obtained from the rhizome of Anemarrhena asphodeloides (AA). For more than a decade, studies on the anticancer effects of TS-AIII have been reported, and it has been shown that TS-AIII exerts its anticancer effects in a variety of tumor cells mainly by inducing apoptosis and cell cycle arrest through multiple pathways (72). In terms of antitumor drug resistance, related studies have shown that either TS-AIII or AA treatment could significantly inhibit growth and promote cell cycle arrest in PANC-1 and BxPC-3 cells (pancreatic cancer cells with varying degrees of resistance to gemcitabine). Furthermore, when used separately in combination with gemcitabine, both TS-AIII and AA induced caspase-dependent apoptosis of pancreatic cancer cells more than gemcitabine alone. The underlying mechanism may be related to the regulation of the activity of PI3K/AKT pathway proteins involved in the cell cycle and proliferation (73). Another study demonstrated that TS-AIII could inhibit cell growth and induce apoptosis in paclitaxel-resistant tumor cells (A549/Taxol and A2780/Taxol) by suppressing activation of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK signaling pathways, resulting in a more stable antitumor effect (74).

As one of the ‘vulnerable’ species designated by the International Union for Conservation of Nature Red List, Paris polyphylla is an important medicinal plant in the traditional system of medicine, and steroidal saponins are one of the main bioactive chemical components of this plant (75). Polyphyllin I (PP-I) (Fig. 2B) and PP-II (Fig. 2C) isolated from the rhizome of Paris polyphylla, have been proven to have an obvious effect on reversing tumor drug resistance. In a previous study (76), the overexpression of the long noncoding RNA metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) increased signal transducer and activator of transcription 3 (STAT3) expression in NSCLC cells, leading to gefitinib resistance in the lung cancer cells, whereas PP-I downregulated the expression of MALAT1, inhibiting the phosphorylation of STAT3 and finally leading to the apoptosis of NSCLC cells. Similarly, PP-II triggered apoptosis to strengthen the sensitivity of PC-9/ZD cell lines to gefitinib by downregulating the protein levels of PI3K, AKT and mTOR, and upregulating the levels of Bax, caspase-9 and caspase-3 (77).

N45 (Fig. 2D), a steroidal saponin once known as saponin 9 is derived from the rhizome of Paris vietnamensis (Takht.). Liu et al (78) showed that N45 exhibited significant cytotoxic effects on glioblastoma cells with IC₅₀ values of 3.14 µM in U251 cells and 2.97 µM in U87MG cells. The same group proved that N45 could induce mitochondrial apoptosis to inhibit the proliferation of temozolomide-resistant glioblastoma cells (U87R) by increasing the Bax/Bcl-2 ratio, and the levels of cytochrome c and cleaved caspase. The underlying molecular mechanism included the reactive oxygen species (ROS)-mediated inactivation of the PI3K/AKT pathway, which resulted in downregulated expression of the nuclear factor-κB (NF-κB) p65 and O6-methylguanine-DNA methyltransferase (79).

Similarly, by triggering the apoptotic pathway, steroidal saponins can work synergistically with chemotherapy drugs to enhance the sensitivity of tumors to chemotherapy. Paris saponin I (PS-I) (Fig. 2E), isolated from Paris polyphylla, has been reported to induce apoptosis by regulating the expression of Bcl-2, Bax and caspase-3 proteins, and by promoting G₂/M phase cell cycle arrest by activating P21^waf1/cip1 when combined with cisplatin, thereby improving the sensitivity of gastric cancer cell lines to cisplatin (80). In lung cancer cell lines, PS-I similarly acted as a chemosensitizer of camptothecin (CPT)/10-hydroxycamptothecin (HCPT), which synergistically inhibited cell proliferation and induced apoptosis by improving activation of the p38 MAPK/caspase signaling pathway in H1299 cells, as well as by inhibiting activation of the AKT and ERK pathways in H460 and H446 cells (81). Combination treatment with ginsenoside Rh2 (Fig. 2F) and cisplatin has been shown to potentially overcome the tolerance of NSCLC cells to cisplatin by promoting apoptosis and inhibiting cisplatin-induced phosphorylation of EGFR, PI3K and AKT, thus suppressing the production of superoxide, the expression of programmed death-ligand 1 and autophagy (82).

According to in vitro and in vivo research, autophagy induced by doxorubicin in hepatocellular carcinoma cells promoted tumor cell survival. By contrast, the 20(S)-ginsenoside Rg3 (G-Rg3), a stereoisomer of G-Rg3 (Fig. 2G), which was isolated from steamed Panax ginseng C.A. Meyer, was shown to suppress the late stage of autophagy by inhibiting the maturation, fusion or degradation stages, thus exhibiting a positive effect on doxorubicin-induced hepatocellular carcinoma cell death. The potential mechanism of this effect was partly relevant to regulation of the C/EBP homologous protein (CHOP) transcription factor at the genomic level (83). Besides, the combination of G-Rg3 and paclitaxel was able to promote cytotoxicity and apoptosis of triple-negative breast cancer cell lines by interrupting the NF-κB signaling pathway, thus downregulating the protein levels of NF-κB, p65 and Bcl-2, and upregulating the levels of Bax and caspase-3 (84). An earlier study also showed that G-Rg3 could enhance the antitumor effects of radiation therapy on NSCLC cells by targeting and regulating the NF-κB protein and its regulatory gene products (85).

In addition, a series of findings made by Wang's research team with regard to black nightshade (Solanum nigrum L.) found that the promotion of autophagy could significantly inhibit the proliferation of drug-resistant tumor cells. Solanum nigrum L. is a plant belonging to the Solanaceae family that commonly grows in Africa and Southeast Asia, and has been widely used as a vegetable, fruit and source of various therapeutic medicines for a number of years (86). Wang et al (87) found that S-20 (Fig. 2H), a novel component isolated from the berries of black nightshade, induced both autophagy and caspase-dependent apoptosis to overcome the Adriamycin resistance of K562 cells (K562/ADR); however, upon the addition of inhibitors to these two pathways, it was discovered that autophagic death was the primary pathway through which S-20 exerted its anti-drug resistance effect, rather than apoptosis. The mechanism of action was associated with the activation of ERK, which further suppressed the expression of BCRP and P-gp proteins (87). By contrast, in a subsequent study, this research group reported that the total saponins from the berries of Solanum nigrum exerted anti-drug resistance activity by significantly downregulating the phosphorylation level of mTOR kinase in K562/ADR cells and xenograft tumors, inducing autophagy in K562/ADR cells and inhibiting the expression of drug resistance proteins (88). Furthermore, the synergistic combination of the total saponins of Solanum nigrum and Adriamycin could induce apoptosis through the intrinsic and extrinsic pathways, and activate autophagy by downregulating the PI3K/AKT/mTOR signaling pathway and upregulating the MAPK signaling pathway, thereby significantly enhancing the antitumor resistance activity in K562/ADR cells (89).

PP-I has been demonstrated to resensitize HCC827-ER cells to erlotinib and to enhance antitumor activity by reversing the EMT process through inhibitory effects on the activation of the IL-6-mediated signaling pathway and the phosphorylation of STAT3 protein, thereby decreasing the levels of vimentin and increasing those of E-cadherin (90). Furthermore, PP-I combined with polyphyllin VII (PP-VII) (Fig. 2I) could inhibit the invasion and metastasis of cisplatin-resistant NSCLC cells (A549/DDP) by upregulating levels of the epithelial marker E-cadherin, and downregulating those of the mesenchymal markers vimentin and α-smooth muscle actin. Meanwhile, the combination also induced apoptosis and autophagy to promote A549/DDP cell death via upregulation of p53 expression and inhibition of the cancerous inhibitor of protein phosphatase 2A/AKT/mTOR signaling axis (91).

A study on the inhibitory effects of diosgenin (DG) (Fig. 2J) on breast cancer stem cells (bCSCs) showed that DG induced apoptosis by activating caspase-3/7 and releasing ROS. Further investigation identified that, in sFRP4-OE cells, a model of bCSCs that overexpressed Wnt antagonists, DG treatment significantly increased the expression of E-cadherin, decreased N-cadherin and β-catenin proteins, and downregulated the expression of the pro-invasive genes Twist and Snail, thus inhibiting EMT and suppressing the invasiveness of bCSCs, probably via the Wnt/β-catenin pathway (92).

In human chronic myelogenous leukemia Adriamycin-resistant cells (K562/ADM), TS-AIII exhibited the ability to downregulate overexpressed P-gp and MRP1 in a dose-dependent manner, further improving the retention of Adriamycin in the cells, and the underlying mechanism may be related to the PI3K/AKT signaling pathway in this process (93). Bufalin (Fig. 2K), an extract of the natural Chinese herbal medicine Venenum bufonis, has been reported to prevent Adriamycin outflow by inhibiting nuclear factor erythroid 2-related factor 2 and weakening the expression of the downstream target genes, including heme oxygenase-1 and P-gp, thus reversing the drug resistance of K562/A02 cells (94).

Trillium tschonoskii Maxim (TTM) is a folk medicine that originated from Liliaceae in China. TTM has long been known as ‘Yan Ling Cao’ and is used to treat traumatic brain injury and headaches (95). Previous studies showed that the steroidal saponin of Trillium tschonoskii (TTS) could downregulate the expression of P-gp in R-HepG2 (a cell line in which the sensitivity to doxorubicin was much lower than that of parental cells) in a dose-dependent manner at both the transcriptome and protein levels (96). Moreover, TTS treatment enhanced the cytotoxic effect of doxorubicin on primary tumors both in vitro and in vivo. Paris saponin VII (PS-VII) (Fig. 2L) derived from the roots of TTM has been reported to reduce Adriamycin transmembrane outflow in Adriamycin-resistant breast cancer cells by inhibiting the expression and function of P-gp at a low dose (97). Another study demonstrated that PS-VII significantly enhanced the sensitivity of HepG2 cells to Adriamycin via inhibition of the PI3K/AKT/MAPK signaling pathway, thus decreasing the expression of P-gp, MRP1 and BCRP proteins, increasing the intracellular accumulation of Adriamycin and also inducing cell apoptosis (98). Additionally, co-incubation of H1975 cells with PP-VII and gefitinib enhanced the anti-proliferative effect of gefitinib by upregulating p21 protein expression, and downregulating the expression levels of cyclin-dependent kinase (CDK)2, CDK4, Cyclin E and Cyclin D1, leading to a cellular G₁-phase block (99).

Low cytotoxic concentrations of total saponins from Paris forrestii inhibited ERK phosphorylation through the MAPK signaling pathway, thereby reducing expression of MDR1 mRNA and P-gp protein, ultimately reversing drug resistance in Adriamycin-resistant human breast cancer cells (MCF-7/ADM) (100).

Shenmai injection (SMI) is derived from the famous Chinese patent medicine known as Shenmai San, which has been clinically used for the treatment of cardiovascular and cerebrovascular diseases. A previous study demonstrated that SMI could inhibit the function and expression of P-gp through the MAPK/NF-κB signaling pathway, and further potentiate the sensitivity of breast cancer cells to chemotherapeutic drugs (101). Moreover, Panax ginseng and Ophiopogon japonicus, as the primary components of SMI (102), have been proven to be rich in steroidal saponins (103,104). Based on the aforementioned facts, the present review indicates the potential possibility to further investigate the effects of SMI components on the reversal of tumor drug resistance.