Salinomycin, as an autophagy modulator-- a new avenue to anticancer: a review

According to the latest World Health Organization (WHO) data, cancer is the second-leading cause of death globally and accounts for 8.8 million death in 2015 [1]. However, major current tumor therapeutic strategies like operation, radio- and chemo-therapy still exist some defects, failing to cure most tumor patients completely. Cancer stem cells (CSCs), which are resistant to many current anticancer therapies, perhaps account for the failure of treatments. CSCs refer to the subpopulation of cancer cells endowed with self-renewal, multi-lineage differential capacity and innate resistance to conventional radio- and chemo- therapy [2]. CSCs, relying on those capacities, are regarded as the culprit of recurrence and metastasis of cancer [3, 4]. Hence, eradication of CSCs will be the key to the success of cancer treatment.

Of note, with further study of Sal, it stands out as one of the notable landmarks in the progress of chemotherapeutical drugs on CSCs. Sal, isolated from the bacterium Streptomyces albusin 1974 [5](See Fig. 1), exhibits a broad-spectrum antibiotic activity particularly against Gram-positive bacteria, fungi, parasites, protozoa [5, 6]. It is widely used as an anticoccidial drug in animal farming and is fed to ruminants to improve nutrient absorption and promote growth [7]. In 2009, Gupta et al. screened about 16,000 compounds in order to hunt for chemicals that are preferentially toxic to CSCs. The screening identified 32 substances that are able to impair CSCs. Finally, Sal was found to be the most efficient one, having a more than 100-fold efficiency of Sal compared to Paclitaxel to kill breast CSCs in mice [8]. After that, the efficiency of Sal against the CSCs in several malignancies, including breast-, prostrate-, brain-, blood-, liver-, pancreatic-, skeleton- and lung cancers have been further verified [9‐12]. In addition, it has been proved that Sal is able to kill chemotherapeutical agents resistant cancer cells such as Doxorubicin-, Cisplatin-, Gemcitabine-, Temozolamide-, verapamil- and Imatinib- resistant cells and simultaneously sensitize radio-resistant cancer cells [9, 13‐15]. Besides its predominant anticancer activities, it has been also verified that Sal does not emerge severe adverse effects on human normal tissues like other conventional chemotherapeutical drugs. Sal induces T-cells apoptosis in T-lymphocytic leukemia patients, but not in healthy people [16]. Similar results have been demonstrated in further studies [17, 18]. Furthermore, several successful pilot studies in cancer patients have showed temporary and minor effects while causing the regression of various solid tumors [9, 19].

Despite the predominant antitumor effects and fewer adverse effects of Sal, the mechanism by which Sal brings about cancer cell death while non-malignant cells are exempted from the lethal effects remaining poorly understood. According to the studies both in vivo and in vitro, such following mechanisms that mitochondria-dependent cell death [20, 21], Death receptor - mediated cell death [14], increased DNA damage and cell cycle arrest [22, 23], p-glycoprotein inhibition [24, 25] have been reported to involve the predominant anticancer effects of Sal. However, those mechanisms may just play a partial role in the anti-cancer effects of Sal as conventional chemotherapeutical agents rely on them to induce cancer cell death. Further studies have demonstrated that Sal suppresses Wnt/β-catenin signaling pathway conferring CSCs resistance to radiation [26, 27] and to chemotherapeutical agents [28, 29]. Moreover, other studies have showed that Sal blocks the Hedgehog (Hh) pathway, which plays a crucial role in the stemness maintenance of CSCs [30, 31]. Those may elucidate Sal’s preferential toxicity towards CSCs. Besides the classical cell death pathway, alternative cell death pathway has been also observed in the experiments, the result has displayed that Sal is able to mediate apoptosis in cancer cells through a pathway independent of activation of p53, CD95/CD95L system, caspase, and the proteasome [32]. A variety of cell death pathways or the apoptotic signal pathways forming a complex reticular structure involves in the anticancer effects of Sal, but the key component to initiate that process is still unknown, what a role autophagy plays in the reticular structure requires more studies.

Conventional chemotherapeutical drugs cannot efficiently kill CSCs, which led to the recurrence of the cancer [4, 33, 34]. Comparing to the conventional chemotherapeutical drugs, Sal targeting CSCs must have its unique targets or signaling pathways. Therefore, the study of Sal cannot only provide an effective tumor treatment drug, but also can deepen the understanding of the characteristics of CSCs, so as to give a basis for the development of new anticancer drugs, and ultimately overcome this catastrophic disease.

With a further research, scientists have found that autophagy is a homeostatic process that prevents the accumulation of impaired proteins and organelles and limits the production of reactive oxygen species (ROS). Autophagy can simultaneously provide the cell with nutrients and energy that it needs to survive. However, autophagy plays multifarious roles in the initiation, development and progression of cancer. On the one hand, autophagy suppresses malignant transformation by many mechanisms such as maintaining genomic stability via the elimination of dysfunctional mitochondria and the decrease of ROS production, the degradation of onco-proteins, the induction cell death following oncogene activation, anti-inflammatory function and assisting the immune system to eliminate the malignant cells [35]. On the other hand, after a formation of neoplastic lesion, autophagy is believed to promote and maintain tumorigenesis and to mediate resistance to different forms of anticancer therapy [36]. Autophagy is generally considered to play a cyto-protective role for cell survival against apoptosis. However with further study, many literatures have demonstrated that autophagy is primarily a cell survival pathway, but if excessive, it may kill cells [37‐39]. So the exact role of autophagy playing in the death process of cancer cells requires further study. Additionally, autophagy is dysregulated in various diseases, such as neurodegenerative diseases, immune system diseases and cancers.

Chemotherapeutics currently used kill tumor cells but also cause impairment to human normal tissues, such as myelosuppression, immunosuppression, nephrotoxicity, liver injury. Interestingly, Sal selectively induces cancer cells death while sparing benign cells. This may be related to Sal’s distinct targets or signaling pathways that are lethal to cancer cells while are slighter to human normal tissues. Here we will explore whether the inhibition of autophagy is one of the explanations for Sal’s preference for cancer.

Mitochondria, generally called the powerhouses of the cell, comprises a dynamic interconnected network of tubular structures that are engaged in fission and fusion processes [55]. Mitochondrial fission is mediated by localization of dynamin-related protein (Drp-1) on the mitochondrial site of division whereas fusion is mediated by mitofusin proteins (mitofusins 1 and 2) along with optic atrophy protein 1 (OPA1) [56]. Lack of both mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) results in damaged mitochondrial fusion, but both of them can compensate each other’s deficiency [57‐60]. Facing stress or any event leading to dysfunction of mitochondria, the organelle undergoes an asymmetric and protective fission that aims to spare at least part of stressed mitochondria, by cleaving the organelle into a normally-functional part, and a dysfunctional one. A recent study has described that cancer cells often have much smaller, fragmented mitochondria in comparison to normal cells. In the same study, the results have showed that cancer cells express an increased level of Drp1 and decreased level of Mfn protein, leading to a constant mitochondrial fission with impaired fusion, resulting in a smaller fragmented mitochondria in cancer cells [61]. Another study has displayed that specific toxicity of Sal to cancer cells is through mitochondrial hyperpolarization that is observed preferentially in cancer cells. Moreover, Sal-triggered depletion of cellular ATP, in cancer cells but not in primary cells, contributes to Sal’s preferential anticancer toxicity to cancer [62]. Furthermore, Sal induces depolarization of mitochondria contrasting to Dichloroacetate (DCA), which is also a K⁺ ion channel modulator, and a molecule that also preferentially targets cancer cells [63, 64]. The observed cancer cells specific deleterious effect of Sal is explained by the fact that cancer cells harbor various abnormalities within mitochondria, such as hyperpolarized mitochondria along with defective mitochondrial fission and fusion mechanisms due to the lack of functionally intact mitofusin proteins, DRP1 and other proteins involved in the mitochondrial dynamics [61, 63]. Similarly, a recent study has pointed out that Sal in concentrations effective against CSCs exerts profound toxicity towards both dorsal root ganglia as well as Schwann cells. With further study, the result has showed that the toxic effect is mediated by elevated cytosolic Na⁺ concentrations, which in turn causes an increase of cytosolic Ca²⁺ by means of Na⁺/Ca²⁺ exchangers (NCXs) in the plasma membrane as well as the mitochondria membrane. Elevated Ca²⁺ then activates calpain, which triggers caspase-dependent apoptosis involving caspases-12, − 9 and − 3. Combined inhibition of calpain and the mitochondrial NCXs result in significantly decreased cytotoxicity as compared to caspase-3 inhibition in vitro [65]. Furthermore, co-treatment with an inhibitor of the mitochondrial NCX can significantly reduce structural damage in the peripheral nervous system without impairing Sal’s antineoplastic efficacy in vivo [66]. This may account for the abnormal mitochondria in cancer cells, which is not resistant to the effect of Sal. Fig. 3 shows the conceivable mechanism by which Sal damages peripheral nervous system.

Emerging data indicate the crucial role of autophagy in the survival, self-renewal and differentiation of both stem cells and CSCs [67‐69]. Recent study has found that the basal autophagic flux is higher in the ALDH(+) population derived from the HMLER cell line than in the corresponding ALDH1(−) population, the former owns more CSCs features than the latter. Autophagic flux falls in both ALDH(+) and ALDH(−) populations following Sal treatment. However, the fall of autophagic flux is greater in ALDH(+) population than in the ALDH(−) one (51% vs. 10%, respectively). Consistent with data has showed that apoptotic cell death was higher in the ALDH1(+) population derived from the HMLER cell line than in the ALDH(−) population [45]. Similar results have been acquired by another study that the subline HMLER CD24 low, carrying CSC properties has an increased sensitivity to autophagy inhibition mediated by Sal with respect to the non-CSC subline HMLER CD24(+) [52].

Autophagy is generally considered to be vital for the elimination of dysfunctional mitochondria that may actually consume ATP, and/or generate excessive amounts of harmful ROS, thus contributing to loss of cellular homeostasis [70], thus the inhibition of autophagy will enhance the apoptosis. The elimination of dysfunctional mitochondria is highly dependent on autophagy, however, Sal can induce a profound inhibition on the late stage of autophagic flux. That is, on the one hand to induce deformed mitochondria in cancer cells to be dysfunctional, on the other hand Sal inhibits the elimination of these dysfunctional mitochondria with increased ROS-production and induction of apoptosis.

Since the first article, reporting that Sal is able to induce autophagy in cancer cells, was published in 2012 [40], different tumor cell lines have been used to detect the mechanism of autophagy induced by Sal. Sal is a K⁺ ionophore that interferes with transmembrane K⁺ potential and facilitates the efflux of K⁺ from mitochondria [71]. Sal causes the decline of mitochondrial membrane potential (MMP) and the accumulation of ROS [20, 72]. Most of the tumor cells are found to own increased ROS after the treatment with Sal, which plays an important and complex role in autophagic activation. To clarify the role of ROS in Sal-induced autophagy, U2OS cells are pre-treated with N-acetyl-L-cysteine (NAC), a ROS inhibitor. As a result, the sharp decrease of LC3II expression and acidic vesicular organelles (AVO) accumulation is detected [20]. Similar results are also found in osteoblastoma [42], breast cancer [73], prostate cancer [74], glioma [75]. Growing data have manifested that ROS inhibits PI3K/AKT/mTOR signaling [76] and activates the AMP-activated protein kinase (AMPK) signaling [77]. ROS is also reported to initiate mitogen-activated protein kinase (MAPK) signaling, including of C-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) [78] (See Fig. 4). Except for the three mechanisms mentioned above, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is also considered to be required for Sal-induced autophagy activation [18]. Table 1 has summarized the relevant information in recent advances.

It has been reported that Sal also induces apoptosis, which differs among the diverse cell types, in cancer cells [14, 16, 72, 103, 104] or different originated CSCs [7, 21, 105]. Apoptosis is induced in colorectal CSCs in a caspase-dependent manner, which proved by an increase of cleaved caspase-3, -8 and -9. JC-1 staining further revealed that Sal induces colorectal cancer cell apoptosis via the mitochondrial pathway [21]. Besides, Sal has been reported to induce human lung cancer cell apoptosis through the caspase 3/7-associated cell death pathway [106]. Moreover, Sal triggers apoptosis of PC-3 cells by increasing the intracellular ROS level, which is associated with reduced MMP, translocation of Bax protein to mitochondria, release of cytochrome c to the cytoplasm, activation of the caspase-3, cleavage of PARP-1 as well as the inhibition of the survival protein Bcl-2 [72]. It also has been noted that non-malignant RWPE-1 prostate cells are relatively less sensitive to Sal-induced lethality [72]. Besides the classical apoptotic pathway, Sal has been demonstrated to activate a unique apoptotic pathway that is not accompanied by cell cycle arrest and is independent of tumor suppressor protein p53, caspase activation, the CD95/CD95L system and the proteasome, which has been thought to overcome the high resistance to other anti-cancer drugs of cancer cells by increasing the expression of the Bcl-2 protein, P-gp as well as the 26S proteasome [16].

Uncontrolled cell cycle is an extremely important part of tumorigenesis. Several studies have also found that Sal can play a role in inhibiting cancer cell proliferation by inducing cell cycle arrest. It has been documented that Sal induces apoptosis in NCI/ADR-RES, DXR, and OVCAR-8 ovarian cancer cells via downregulation of S-phase kinase-associated protein-2 (Skp-2) and signal transducer and activator of transcription 3 (Stat3) inactivation. Skp-2 is overexpressed in majority of the CSCs. p27kip1 protein inhibits tumorigenesis through inhibiting cell cycle changes. Sal induces degradation of Skp2 and thus accumulated p27Kip1, ultimately cell cycle being arrested at G1 and apoptosis being induced [25]. Another study has showed that high concentrations of Sal induces a G2 arrest via downregulation cyclin B1, downregulation of survivin and triggering apoptosis. What is an interesting thing is that the administration with low concentrations of Sal induces a transient G1 arrest through downregulation cyclin D1 at an earlier time point and G2 arrest via downregulation cyclin B1 at a later point [107].

Autophagic cell death (ACD), named type II programmed cell death, distinguishes from apoptosis and necrosis, which was first established based on observations of increased autophagic markers in dying cells [108]. There is a big difference between ACD and apoptosis process. The morphological characteristics of apoptosis-related cells are the clearance of cytoskeleton and other proteins by caspases. Cytoskeletal clearance occurs in the early stages of apoptosis, but the organelles are cleared later; On the contrary, ACD is associated with a large accumulation of autophagic vacuoles in the cell, so that the degradation of organelles occurs at an early stage, and the cytoskeleton is cleared at a later stage [109]. In most cases, autophagy can serve as a cell survival promoter, however, autophagy may also act as cell death inducer. The pro-apoptotic effect of autophagy may result from two independent functions of autophagy: the pro-apoptotic function and the induction of ACD [108]. The autophagy regulator Atg5 activates caspase by interacting with the adaptor protein Fas-associated protein with death domain (FADD), a component of the extrinsic apoptotic pathway. Alternatively, cleavage of Atg5 by calpain causes the truncated Atg5 protein to translocate to the mitochondria to induce mitochondrial outer membrane permeabilization (MOMP) leading to cytochrome c release and caspase activation [109]. As a whole, there is a complicated relationship between autophagy and apoptosis. As mentioned above, Sal has been reported to induce cell death mainly via mitochondria-mediated apoptotic pathway, which can be considered as the executor of cancer cell death resulting from the severe damage caused by Sal. Whether the conjecture that the abnormal mitochondria in cancer cells or CSCs, which are not resistant to the effect of Sal, can be impaired by Sal and thus initiates the cell death process are true or not being worthy of further study. Moreover, several studies have showed that Sal both induces intensive autophagy and inhibits the late stage of autophagic flux which may play a crucial role in the ACD or pro-apoptotic process being of equal significance.

Sal, being a K⁺ ionophore, can effectively target CSCs, kill chemo-resistant cancer cells and sensitize radio-resistant cancer cells simultaneously. Of note, its side effects are much less than the conventional chemotherapeutical drugs. Although Sal exhibits predominant anticancer effects in vivo and in vitro experiments and a small number of clinical trials, the exact mechanisms are still unknown.

We summarize the autophagy-related researches, providing a new sight into Sal’s preference for CSCs and cancer cells. Cancer cells are deemed to possess abnormal mitochondria, which may be not resistant to ion fluctuations caused by Sal, then leading to the dysfunction of organelles and the production of ROS, which initiates autophagy. Similarly, DCA, which is likewise a K⁺ ion channel modulator, preferentially targets cancer cells. Meanwhile, Sal can inhibit the late stage of the autophagic flux, which leads the accumulation of dysfunctional organelles and produces excessive ROS. The combined effects of these two processes lead to the death of tumor cells. The combination of a K⁺ ionophore and autophagic inhibitor may be able to confirm the rationality of this conjecture.

In addition, some studies have showed that the combined use of Sal and CQ, which is an autophagy inhibitor, not only improves antitumor effects of Sal but also reduces the dose of Sal [44]. Whereas, Sal is able to inhibit the late stage of autophagic flux, so whether the combined use of an early stage inhibitor or a late stage inhibitor can amplify the anticancer effects of Sal requires further studies. What’s more, another study has found that co-treatment with an inhibitor of the mitochondrial NCX can significantly reduce structural damage in the peripheral nervous system without impairing Sal’s anticancer efficacy in vivo [65]. Therefore, a large number of further studies should be carried out to provide a basis for future rational combinative therapy.

CSCs are generally considered to be the chief culprit for the initiation, recurrence and metastasis of cancer. Sal preferentially targeting CSCs may bring us closer to find a cure against this devastating disease. Generally speaking, specific and more potent toxicity of Sal towards CSCs and cancer cells without much adverse effect towards normal cells promise Sal may be an effective anticancer agent in the near future. Understanding the biological mechanism of Sal inducing cell death can aid in designing more effective and less toxic therapeutic strategies. As a new anticancer compound, whether Sal could eventually enter into clinical application needs the joint efforts of all researchers. Anyway the in-depth study of Sal’s direct targets or related signaling pathways will open a new chapter in the near future against cancers.