Anlotinib, an inhibitor of multiple tyrosine kinase receptors, inhibits tumour progression by inhibiting angiogenesis (Han et al.,2018), but there is no published literature on the inhibitory effect of anlotinib on BC.
Many studies have reported that anlotinib exerts antitumour effects to inhibit cell viability and proliferation in hepatocellular carcinoma (HCC), lung cancer, thyroid cancer and osteosarcoma (He, Wu, & Hao,2018;Liang et al.,2019;Ruan et al.,2019a;Wang et al.,2019). Our study found that anlotinib also inhibited the cell viability and proliferation of BC cells, which was consistent with the effects in other tumours. Cell proliferation is regulated by the cell cycle, and Cyclin is an important regulatory molecule that drives the cell cycle. Cyclin D1 is a member of the cyclin family that is activated in the G1 phase and drives G1/S phase transitions. Cyclin D1 binds to cyclin-dependent kinase 4 and cyclin-dependent kinase 6, induces excessive retinoblastoma protein phosphorylation, thereby shortening the G1 phase, promoting excessive cell proliferation and leading to tumourigenesis(Ahlin et al.,2017). This study found that the expression of Cyclin D1 mRNA and protein in BC cells decreased significantly after anlotinib treatment. In contrast, the G1 phase decreased unexpectedly. It is possible that the increased G2/M phase and specific and deeper mechanisms require further investigation. The above evidence suggests that anlotinib inhibits the proliferation of BC cells by prolonging the cell cycle. Similar to our results, anlotinib in thyroid cancer causes abnormal spindle assembly and G2/M arrest, inhibiting cell cycle progression (Ruan et al.,2019b).
Autophagy is an important cellular mechanism that plays a "housekeeping" role in normal physiological processes, including the removal of longevity, aggregation and misfolded proteins, removal of damaged organelles, and the regulation of growth and ageing. In tumour cells, autophagy is usually activated during anticancer treatments such as radiation therapy, chemotherapy, and targeted therapy. This may be a cytoprotective mechanism that also causes excessive autophagy in the cell, namely, excessive self-digestion, and induces phagocytic cell death, which is also known as type II programmed cell death (Ravanan, Srikumar, & Talwar,2017). A study found that anlotinib induced autophagy in human lung cancer cells in a time- and concentration-dependent manner and increased the ratio of LC3BII/I protein and the protein expression level of BECN1. Using the autophagy inhibitors 3-MA and BECN1, small interfering RNA reversed the autophagy effect induced by anlotinib; unexpectedly, it enhanced the inhibitory effect of anlotinib on cell proliferation, making the anticancer effect of anlotinib more sensitive and strengthening its inhibition of angiogenesis (Liang et al.,2019). This suggests that the induction of autophagy in human lung cancer cells by anlotinib is a cytoprotective effect. In our study, anlotinib also induced autophagy in MCF-7 and MDA-MB-231 human BC cells. These results showed significantly increased mRNA expression levels of LC3B and BECN1 and the ratio of LC3BII/I protein and BECN1 protein levels and decreased P62 protein levels, suggesting that promoting autophagic cell death may be an important mechanism by which anlotinib inhibits BC cell growth.
Apoptosis is a common programmed cell death and plays a key role in the development of diseases, including cancer. Cancer cells evade apoptosis, thereby achieving excessive proliferation and surviving under hypoxic conditions and with drug resistance (Matsuura, Canfield, Feng, & Kurokawa,2016) ; thus, promoting tumour cell apoptosis has become an important strategy for the treatment of cancer. Studies have shown that anlotinib exerts its antitumour effects on HCC, thyroid cancer, osteosarcoma and lung cancer by promoting apoptosis (He et al.,2018; Liang et al.,2019;Ruan et al.,2019a;Sun et al.,2018;Wang et al.,2019). Anlotinib significantly inhibited colony formation and promoted apoptosis in HCC and thyroid cancer in vitro (He et al.,2018;Ruan et al.,2019a). It upregulated the pro-apoptotic molecule Bax and inhibited the anti-apoptotic proteins Bcl-2 and Survivin to kill tumour cells. In addition, animal experiments demonstrated that anlotinib reduced the volumes and weights of transplanted tumours (He et al.,2018). In thyroid cancer, anlotinib caused abnormal spindle assembly and G2/M arrest, promoted the activation of cleaved-Caspase 3 and cleaved PARP, and activated TP53 (Ruan et al.,2019a). Similar to the above experimental results, we found that anlotinib increased the mRNA and protein levels of proapoptotic proteins and inhibited the mRNA and protein levels of the anti-apoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 BC cells, thereby exerting an antitumour effect.
Autophagy and apoptosis often occur in the same cells with the same upstream cellular signals activated by the endoplasmic reticulum, such as extracellular regulated protein kinases (ERK)/ activating transcription factor 4 (ATF4), Inositol-requiring enzyme-1α, ATF6, and Ca2+. On the one hand, autophagy not only blocks the induction of apoptosis by inhibiting the activation of apoptosis-associated caspases and reduces cell damage but also induces apoptosis. On the other hand, activation of apoptosis-related proteins also suppresses autophagy by degrading autophagy-related proteins such as BECN1, autophagy-related protein 4D (ATG4D), ATG3 and ATG5, but the specific mechanisms of their mutual regulation in BC cells need further study (Song, Tan, Miao, Li, & Zhang,2017). However, in our study, we utilized the autophagy inhibitors wort and 3-MA before anlotinib treatment and found that inhibition of autophagy reversed anlotinib-induced apoptosis in BC cells. We obtained the opposite results compared with that reported in lung cancer cells (Liang et al.,2019). This is probably due to the different concentrations of autophagy inhibitors and different cells, but it remains unclear and needs further study to clarify the crosstalk between apoptosis and autophagy.
Evidence indicates that Akt is a key molecule in both autophagy and apoptosis because it is the upstream signal of mammalian target of rapamycin complex and JNK (Heras-Sandoval, Pérez-Rojas, Hernández-Damián, & Pedraza-Chaverri,2014;Yu et al.,2017;Zhang et al.,2018). It is also a downstream protein of VEGFR signalling. Previous studies reported that anlotinib inhibits Erk and Akt signal transduction pathways to regulate cell growth in HCC cells (He et al.,2018). Hence, we investigated Akt signalling and found that inactivated Akt/GSK-3α signalling in anlotinib-induced apoptosis was reversed by autophagy inhibitors, suggesting that anlotinib-induced autophagy promotes apoptosis by impacting Akt/GSK-3α signalling.
In summary, our study demonstrated that anlotinib inhibited the growth of BC cells via promoting apoptosis through autophagy mediated by Akt/GSK-3α signalling and may be an effective new drug for BC treatment.