Numerous natural Traditional Chinese Medicine (TCM) products exhibit anticancer properties, generally with low toxicity and the ability to protect normal cells(15). Several researchers have validated the inhibition of proliferation in cancer cells by TSPG and other monomers. Our results demonstrate that Rh2 has stronger effects at lower doses than TSPG, Rb1, Re, Rg1, and Rg3 in human leukemia cells(16). It is known that a structure-activity relationship exists among with panaxadiols generally being more active than panaxatriols in killing cancer cells, and ginsenoside compounds being less active as the number of sugar moieties increases(17). Rh2, with a single sugar residue, inhibits various cancer cells and enhances the efficacy of chemotherapy agents when used in combination(18). These results demonstrate that Rh2 has stronger effects due to its special chemical structure. The anti-leukemic effect of Rh2 and its underlying mechanism have not been completely elucidated. In this study, we observed that Rh2 induced growth inhibition and apoptosis in K562 and KG1α cells. Our previous study also illustrated that Rh2 induced apoptosis partially through Caspase3-dependent pathways in K562 and KG1α cells, indicating that one of the underlying mechanisms of the anti-leukemic effect of Rh2 relies on apoptosis.
Apoptosis and autophagy are highly conserved and tightly regulated processes(19). Besides their physiological role in maintaining cellular homeostasis, apoptosis and autophagy are key targets for tumor therapeutics(20, 21). In this study, we investigated whether Rh2 induced autophagy in human leukemic cells. We used three well-established methods to detect autophagosome formation and observed a Rh2-induced autophagic response in K562 and KG1α cells, as evidenced by the formation of autophagosomes using acridine orange and MDC staining, and the increased expression of LC3A/B through western blot analysis. While the induction of LC3A/B on immunoblots indicates increased autophagosomes, it is not sufficient to conclude on the regulation of autophagy flux without investigating cargo degradation(22). The MDC and acridine orange staining results also confirmed that Rh2 promoted autophagy in leukemia cells, consistent with the western blot results.
The role of autophagy in promoting or enhancing survival remains controversial(23). Several researchers have reported that chemotherapy drugs induce autophagic tumor cell death(24). To directly address this question, we investigated the role of autophagy in Rh2-treated human leukemic cells. We pharmacologically inhibited Rh2-induced autophagy using 3-MA in K562 and KG1α cells. Our results showed that Rh2-induced cell apoptosis was significantly reduced with additional treatment of the autophagy inhibitor 3-MA. Furthermore, cell viability analysis demonstrated that the combination of Rh2 and 3-MA enhanced cell vitality more significantly compared to Rh2 treatment alone, indicating that Rh2-induced autophagy might play a cytotoxic role in K562 and KG1α cells.
Our previous studies have shown that inhibits HDAC enzyme activity, reduces HDAC protein expression, and promotes apoptosis of leukemia cells(25, 26). However, the specific mechanism is unknown. HDAC6, which has two enzyme catalytic domains, plays a key role in the transformation of normal cells during cancer and is considered a drug target for anticancer treatment and a prognostic marker for cancer(27). HDAC6 acts as a transcription factor in the cytoplasm and affects the functions of certain non-histone proteins (α-tubulin,HSP90) (28).Autophagy is considered the primary mechanism for clearing protein aggregates(29). To investigate the link between Rh2-induced autophagy and apoptosis, and the role of HDAC6 in the autophagy process, we examined whether HDAC6 properly suppresses autophagy and detected the downstream proteasome protein HSP90. We assessed autophagy activation by first monitoring the upregulated expression of LC3A/B associated with autophagosomes. Unexpectedly, despite the clear defect in protein aggregate clearance, the upregulation of LC3-B is prominently induced in siHDAC6 and Rh2 treatment, suggesting that Rh2 activates autophagosomes normally in the absence of HDAC6. To further evaluate the involvement of autophagosomes in protein aggregate clearance, we examined the changes in proliferation and apoptosis associated with HDAC6 knockdown in leukemia cells (30). The down-regulation of HDAC6 expression increased the likelihood of apoptosis in K562 and KG1α cells. HDAC6 knockdown, which suppresses the expression of Caspase3, is important for apoptosis, activating HSP90 acetylation and causing proteasome-induced apoptosis or autophagy(31). These results indicate that autophagosome formation proceeds normally in the absence of HDAC6. Silencing of HDAC6 activates apoptosis and autophagy by decreasing the expression of HSP90 and the acetylation of HSP90. These results indicate that autophagosome formation proceeds normally in the absence of HDAC6.
Consistent with the previously reported in vitro antileukemic effects of Rh2, the present in vivo study demonstrated that oral administration of Rh2 significantly reduced tumor weights in the Rh2 group compared with the control group. This was further confirmed by the significant depletion of HDAC6 and HSP90 expression in the Rh2 group, consistent with the in vivo findings. Rh2 induced apoptosis and autophagy in leukemia cells, possibly through the inhibition of HDAC6 and the decrease in HSP90 expression in vivo. We further determined that Rh2 could potentially serve as a natural HDAC6 inhibitor for leukemia chemotherapy.
In conclusion, our data demonstrate that Rh2 effectively inhibits the growth of leukemia cells in vitro and in vivo by inducing autophagy mediated by HDAC6. Rh2 inhibits the expression of HDAC6 in leukemia cells, promotes the expression of autophagy and apoptosis-related proteins, and induces programmed cell death (Fig. 7). This study suggests that Rh2-induced cytotoxicity occurs through a novel mechanism and supports its clinical potential as a component of therapeutic strategies for leukemia.