The dried root of Astragalus membranaceus has a long history of medicinal use in TCM. It is an adjunct anticancer agent and it has been the subject of a great deal of research [17, 22, 24]. Studies have shown that APS has anti-tumor activity in vitro when applied alone in certain tumor cell lines, such as murine renal cell carcinoma, murine bladder tumors, HepG2 cells, human gastric cancer SCG-7901 cells, human colon cancer cell lines, hormone-sensitive (MCF-7) breast cancer cell lines, and human hepatocellular carcinoma [13, 17, 24, 25, 29, 30]. Animal tumor models and clinical studies have also confirmed that APS has anti-tumor activity [16, 21–23, 34]. However, there have only been a few reports of the treatment of drug-resistant tumor cells with APS. The present study shows at a final concentration range of 0.8–500 mg/L, the IC50 value of APS for H22/ADM cell proliferation was 251.77 mg/L. According to National Cancer Institute guidelines, extracts with IC50 values < 20 μg/ml are considered active in vitro. The results show that that APS has no anti-tumor activity for H22/ADM cells in vitro when applied alone. However, patients with advanced cancer can be treated with APS combined with chemotherapeutic drugs. It has been found to inhibit tumor development, decrease the toxic-adverse effects of chemotherapy, elevate immune function, and improve patient quality of life [34–36]. For example, Guo et al. reported that treatment with APS injections integrated with vinorelbine and cisplatin significantly improved quality of life in patients with advanced non-small-cell lung cancer over vinorelbine and cisplatin alone . Animal tumor models and in vitro studies confirmed that APS can enhance the chemo-sensitivity of the chemotherapy drugs for non-drug-resistant tumor cells [37–39]. For example, Li et al. reported that the weight of tumors in subjects treated with APS and ADM was significantly lower than those of the NS group . Cui R. et al. reported that hepatocarcinogenesis could be prevented in rats fed with the aqueous extract of Astragalus, which is mainly composed of Astragalus polysaccharides . For H22/ADM resistant cells, as shown in Figure 1 that APS combined with ADM or VCR could significantly reduce the IC50 value (P < 0.05, VS. control group) at APS concentration range of 4 mg/L to 500 mg/L; APS combined with 5-Fu, DDP, VP-16, or CTX could significantly reduce the IC50 value (P < 0.05, VS. control group) at APS concentration range of 0.8–500 mg/L. In this way, APS can enhance the chemo-sensitivity of the most common chemotherapy drugs in vitro. The present results were partially supported by the results of the above-mentioned studies.
Those research teams speculated that the anti-tumor activity of APS might involve enhancement of immune function and induction of apoptosis. However, the mechanism underlying these effects remains to be determined. Changweiqing (Radix Astragali, Radix Codonopsis, Rhizoma Atraety lodis macroce phalae) was found to reverse the drug resistance of colon cancer cells by reducing the expression of MDR1/P-GP . APS is the main active ingredient of Radix Astragali, its involvement in the reversal of MDR merits further investigation [13, 14].
Drug resistance in tumor cells has been shown to be related to MDR1 and P-GP overexpression [40–42]. In the present study, APS was found to enhance the chemo-sensitivity of H22/ADM cell lines to certain drugs. To determine whether APS is involved in P-GP expression and/or its efflux function, the P-GP inducer RFP and P-GP inhibitor VER were used as positive controls, and an H22/ADM group was used as a blank control.
Rh-123 is a cationic dye. It has been used extensively as a marker of P-GP-mediated transport in both in vitro and in vivo studies [43–47]. In the present study, the intracellular fluorescence intensity of Rh-123 increased with increasing concentrations of APS in a concentration-dependent manner in the range of 0.8–500 mg/L. The results show that P-GP efflux activity was inhibited by APS.
Western blot analysis of P-GP expression and quantitative RT-PCR detection of MDR1 mRNA expression in H22/ADM cell lines showed that APS reduced P-GP protein expression and MDR1 mRNA expression in a concentration-dependent manner within the range of 0.8–500 mg/L and in time-dependent manner from 24 h to 72 h. APS not only inhibited P-GP efflux but also reduced P-GP and MDR1 mRNA expression in a concentration-dependent manner. However, this effect was not uniform across all times. For example, APS inhibition of P-GP efflux function did not occur in a time-dependent manner; and there was no visible correlation between time-dependent changes in the MDR1 mRNA levels and corresponding P-GP levels. The modest correlation between mRNA expression and protein abundance in large-scale data sets can be explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Translation is a complicated biological process, and many of the details still merit further investigation. For example, highly expressed proteins may not necessarily require large quantities of mRNA if they have higher than average translation rates. This merits further study .
Drug resistance is a major obstacle to the successful treatment of cancer. Tumor cells either fail to reduce in size following chemotherapy or cancer recurs. The phenomenon of MDR is particularly problematic because it involves the simultaneous resistance to numerous chemotherapeutics of different classes, and the mechanism by which tumors develop MDR is very complex, P-GP overexpression is one important factor in this process . Studies have shown that APS has anti-tumor activity in certain tumor cell lines in vitro and in animal models of certain tumors. The present study confirmed that APS can downregulate MDR1 mRNA expression, inhibit P-GP efflux function and decrease its expression, thereby increasing the intracellular concentration of chemotherapeutic drugs. This may be the mechanism behind its secondary anti-cancer effects.
It has been reported that APS can increase the sensitivity of chemotherapeutics, reducing the side effects and complications associated with chemotherapy, and improve patient quality of life and survival time [12, 14, 19, 20, 23]. In this way, in the search for new cancer therapeutics with minimal toxicity and few side effects, APS is a promising candidate.