Introduction

Cancer is considered the second leading cause of global mortality, with 9.6 million deaths in 2018. This is a combinational disease wherein uncontrolled cell growth occurs and can metastasize to other parts of the body. The mechanisms involved in suppressing tumors in a normal body can differentiate between normal cells and abnormally developing cells. However, the problem arises when genes responsible for tumor suppression get altered by certain environmental factors (including radiation, pollution, and infectious agents) or routine habits of humans (such as alcohol, poor diet, tobacco consumption etc.) [1,2,3,4,5,6,7]. Among the several cancer types, lung cancer and breast cancer account for approximately 11.6% of the total cases prevalent globally, followed by prostate cancer (7.1%) and colorectal cancer (6.1%). Lung cancer has the highest mortality (18.4%), followed by colorectal cancer (9.2%), stomach cancer (8.2%), and liver cancer (8.2%) [8].

Cell death is one of the most significant processes responsible for maintaining homeostasis, by controlling the cell turnover in the body. Based on their biochemical and morphological characteristic, the cellular mortality processes, either due to an inbuilt programmed signaling mechanism or as a result of certain pathological outcomes, are classified into three major categories: (i) autophagy, (ii) apoptosis, and (iii) necrosis [9, 10]. Autophagy is a complex process, and its dysregulation can contribute to the development and progression of cancer. Targeting autophagy can serve as an effective therapeutic strategy in cancer. In the autophagy process, molecular targets have been identified from autophagy induction to lysosomal degradation. The generation of resistance limits the efficiency of current therapeutics (radiotherapy, chemotherapy, immune checkpoint inhibitors, and molecular targeted therapy) in various cancers in response to these therapies.

This poses a need to develop novel therapeutics that can overcome the resistance in a wide variety of cancers and be more effective and safe with low toxicity. For this purpose, natural compounds have drawn the attention of researchers as promising prophylactic and therapeutic strategies for cancer [11, 12].

Celastrol (Cel; Fig. 1) is a quinone methide triterpene present in TWHF root extracts. It is widely recognized as a pharmacologically active compound used in various diseases such as autoimmune, inflammatory diseases, and cancer. The atomic orbital energy analysis reveals that because of the presence of carbon C2 on the A-ring and C6 on the B-ring of celastrol, it is highly susceptible to a nucleophilic attack (Fig. 2) [13]. The quinone methide structure in celastrol has the affinity to react with the thiol groups of the cysteine residues of Cdc37 to form covalent Michael adducts, resulting in the disruption of chaperons or co-chaperones (such as Cdc37-Hsp90 complex) which play a significant role in the stabilization and folding of oncogenic kinases [14]. Some other chaperons/cochaperones proposed to be the target for celastrol in in-vitro studies are p23 [15], IKKβ [16], and the proteasome. This mechanism seems to be one of the major factors responsible for multiple targets of celastrol.

Fig. 1
figure 1

Chemical structure of celastrol [adapted from Ref. [97])

Full size image
Fig. 2
figure 2

The electrophilic sites with positions C2 (Ring A) and C6 (Ring B) in the structure of quinone methide rings (I):-, these sites are susceptible to the nucleophilic attack of thiol groups of cysteine residues to form covalent protein adducts (II) [14]

Full size image

Recent studies have highlighted the potential of celastrol in the treatment of numerous different cancers. Data derived from different animal models and cell lines, attribute the anticancer properties to (i) angiogenesis inhibition, (ii) cell death activation, (iii) anti-invasive effects, and (iv) sensitizing the cells to conventional therapies. Celastrol has been reported to inhibit cancer cell progression and induction of cell death in various cancers such as breast, lung, glioblastoma, hepatoma, nasopharyngeal, prostate, myeloma, colon, pancreas, liver, leukaemia, melanoma, gastric cancer, and osteosarcoma.

Pharmacological activities of natural compounds isolated from Tripterygium wilfordii

Tripterygium wilfordii Hook F (TWHF) is widely known as Thunder of God Vine and has a long history in the treatment of rheumatoid arthritis (RA) [17,18,19]. The root bark of the plant has shown significant pharmacological activities against autoimmune disorders [20], inflammation [21, 22], kidney diseases [23], atherosclerosis, fibrosis, and neurodegeneration [24]. Several bioactive compounds have been isolated from the plant, including sesquiterpenes, glycosides, lignans, alkaloids, diterpenes (triptonide, tripdiolide, and triptolide), and triterpenes (pristimerin, wilforlide A and celastrol) [18, 25, 26]. Of these, celastrol is considered the promising and most active compound of the plant.

Anticancer activities of celastrol

The anticancer potential of celastrol has been widely investigated in vivo in several disease models (Table 1). The development and growth of melanoma xenograft in the mouse models are effectively inhibited by celastrol in a dose-dependent manner [27]. The celastrol treatment has also been shown to suppress the in vitro and in vivo proliferation of bladder cancer cells and osteosarcoma, followed by the induction of autophagy [28, 29]. The viability of HepG2 is inhibited by the disruption of certain signaling pathways when exposed to celastrol alone [30] and affects the expression of EGFR when administered in combination with lapatinib [31]. Treatment with celastrol inhibits the growth of MCF-7 breast cancer cells [32] and causes the induction of apoptosis in HT-29 colon adenocarcinoma cells [33]. The invasion and proliferation of colitis-related colon cancer and NSCLC are suppressed when exposed to celastrol in a dose-dependent manner [34, 35]. Invasion, proliferation, and migration of chondrosarcoma cells are also in vivo [36]. Celastrol stimulates an energy crisis by ATP depletion and induces lipid accumulation, leading to cell cycle arrest and cell death in cancer cells [37]. Celastrol also induces ER stress, leading to growth inhibition of head and neck cancer cells [38].

Table 1 Anticancer activities of celastrol in vivo
Full size table

Signaling pathways associated with celastrol-mediated autophagy regulation

The capability of celastrol to induce autophagy in a variety of cancer cells displays the potential of the compound to modulate multiple signaling pathways (Fig. 3). In various preclinical mouse models, celastrol inhibits the proliferation of tumors by affecting the expression of pro-survival transcription factors and various cell-cycle molecules. Autophagy related markers were identified in cancer cells treated with celastrol and by applying autophagy inhibitors to down-regulate specific markers [39,40,41,42,43,44,45]. In the current review, we focus on summarizing the role of celastrol in cancer therapeutics and giving an overview of the signaling pathways associated with celastrol-mediated autophagy regulation in cancer.

Fig. 3
figure 3

Celastrol-mediated autophagy regulation. The schematic illustrates the main molecular targets of celastrol in the induction of autophagy. Celastrol has multiple targets such as mTOR, ROS, SERCA, miR-17-92a cluster, Atg5 and ERS, thus making celastrol a potential candidate in cancer treatment via autophagy regulation

Full size image

Celastrol induces autophagy via regulation of PI3K/AkT/mTOR pathway

Numerous studies have confirmed the relationship of PI3K, AkT and mTOR pathways with cancer, and their inhibition via autophagy regulation has shown significant results in cancer treatment [46, 47]. These three pathways are linked with each other. AkT was originally identified as an important element in the intracellular signaling of the insulin receptor and is now considered as the significant downstream effector of PI3K activation [48]. PI3K activation results in AkT phosphorylation subsequent to translocation to the inner membrane [49]. The modification of AkT is enough to activate mTOR, which then promotes cell survival and increases protein synthesis by phosphorylating its effectors such as S6K1 and S6K2 [50]. Celastrol has shown a promising role in inducing autophagy by disrupting PI3K/AkT/mTOR pathways (Fig. 4). The disruption of these pathways leads to the autophagy-mediated cell death of cancer cells [27, 51, 52]. The pathways mentioned above are significant for cancer therapy and important for inducing autophagy in the intestine, which could serve as an effective target for treating Crohn's disease (CD) [53].

Fig. 4
figure 4

Celastrol regulates autophagy via disruption of the PI3K/Akt/mTOR pathway

Full size image

Celastrol induces autophagy and promotes G2/M phase arrest via the ROS/JNK signaling pathway

As reported in several studies, ROS generation in excess interferes with various signaling pathways of the cells [54,55,56,57]. Additionally, JNK of the MAPK family plays a pivotal role in regulating autophagy [58,59,60]. Recent studies have highlighted the role of cancer cell survival via synergistic action of JNK with JAK/STAT, NF-κB and other molecules. The pro-survival effect of JNK can be attributed to the immune evasion phenomena mediated by TLR, IFN-γ and TGF-β [61]. Celastrol results in phosphorylation of JNK and increases ROS generation, thereby further promoting autophagy in osteosarcoma cells. Application of ROS inhibitors (such as NAC) reverses the celastrol-induced autophagy and blocks the G2/M phase arrest. However, significant attenuation can be observed when JNK inhibitors are used, but with no impact on G2/M arrest. The phosphorylation of JNK is eliminated by NAC (ROS inhibitor), however, the JNK inhibitor does not affect ROS generation, thereby suggesting ROS as a proximal event for JNK [28].

G2/M is one of the other frontiers serving as a suitable target for anticancer therapy [62]. The cyclin B1 complex promotes the G2/M phase transitions, which remains in the inactivate form by phosphorylation, and the regulation is accomplished by a group of proteins such as Cdc2, Cdc25C, and Chk1/2 [63,64,65]. The expression levels of Chk2, phospho-Chk2, phospho-Cdc2, phospho-Cdc25C, cyclin B1 and p21 are upregulated with celastrol treatment, however, the level of Cdc2 and Cdc25C is downregulated. The level of cyclin B1 is observed to increase with suppression of the Cdc2 activity, which promotes the degradation of cyclin B1 via ubiquitin-dependent proteolysis [66]. The up-regulation of cyclin B1 results in the G2/M phase arrest in cancer cells, thus suppressing cell proliferation [65, 67]. These findings are confirmed by a study that reported that exposure to celastrol inhibits human osteosarcoma’s development and proliferation through autophagy and G2/M arrest. It was also revealed that when the apoptosis was blocked in these cells with suitable inhibitors, the cells died via autophagy; conversely, suppression of autophagy inhibited PARP’s cleavage and caspase-3, thereby leading to apoptotic cell death [28].

Celastrol promotes ER stress/UPR mediated apoptosis and autophagy

In the endoplasmic reticulum (ER) lumen, inappropriately folded proteins accumulate due to internal and external factors in the tumor microenvironment. This accumulation causes ER stress, which results in the activation of Unfolded Protein Response (UPR), an adaptive mechanism for restoring protein homeostasis in the ER. The IRE1α activation and splicing of XBP1 initiate UPR, and these factors are responsible for the transcription of enzymes, particularly chaperons that return to the ER and restore homeostasis. Several studies have linked UPR signaling with different aspects of tumor progression and carcinogenesis [68]. Treatment of different cancer cells (including HCC) with celastrol causes ER stress, with subsequent activation of the UPR for maintaining homeostasis [69]. The proteasome can degrade the unrequired or damaged proteins, but celastrol has an inhibitory effect on proteasome in various cancer cells such as prostate cancer and glioblastoma [70]. In non-functional or disrupted UPR, the homeostasis of protein folding cannot be restored; the persistent stress thereby causes a cascade of events that leads to apoptosis [71].

When the misfolded proteins are not restored or degraded by the proteasome, the UPR mechanism also regulates autophagy [72]. It means that by causing extracellular stress, celastrol not only leads to apoptosis and causes UPR mediated autophagy induction in cancer cells. In HCC, celastrol mediated autophagy was observed through transcription factor of ER stress, and UPR expression induced expression of autophagy-related proteins [73]. However, the direct association between ER stress and celastrol mediated cell death in HCC is not clearly understood and needs further research for understanding the correlation, which will give new insight into the celastrol mediated anticancer effects in HCC [74].

Celastrol induces autophagy by targeting AR, downregulating the miR-17-92a cluster and miR-101

There are some genes and signaling pathways that result in the inhibition of autophagy. One of the important reported gene clusters is miR-17-92a, which exerts a negative role in regulating autophagy. The cluster is transactivated by the androgen receptor (AR) in cancer cells such as prostate cancer [75], and seed sequences have established it as a group of four families: miR-17, miR-18, miR-19, and miR-92 [76]. The dissection of miR-17-92a cluster determined the role of the miR-17 seed family (miR-17 and miR-20) as an autophagy inhibitor in prostate cancer. Another miR-17 family member, known as miR-106, targets the ULK1 to suppress leucine deprivation-induced autophagy in myoblast cells or mycobacteria invasion mediated autophagy [77, 78]. In another study, autophagy in the intestinal epithelial HCT116 cells was inhibited by binding miR-106 to the 30 UTR region of ATG16L [79]. In prostate cancer, several autophagy-related genes serve as suitable targets for the miR-17 seed family, and until now, only the expression of ATG7 is shown to be disrupted when cells are transfected with miR-17 or miR-20a.

Another similar gene (known as miR-101) has also been identified. miR-101 is reported to be an autophagy inhibitor, having a dual role in suppressing both autophagy induction and maturation by targeting the STMN1, RAB5A, and ATG4D genes [80]. However, the AR binding site has been predicted at upstream of the miR-101 gene [81]. Celastrol is highly effective by targeting the AR, promoting the destabilization of AR through inhibition of HSP90, or suppressing calpain activation [82, 83]. Blocking the AR pathway induces autophagy in AR-positive prostate cancer cells [84,85,86,87]. Destabilization of the AR results in suppressing the miR-17-92a cluster and miR-101, subsequently leading to the induction of autophagy in cancer cells [75]. However, the mechanism by which AR regulates autophagy is not fully understood.

Celastrol inhibits SERCA leading to autophagy induction in MDR cancer cells

Some transporter proteins play a significant role in autophagy regulation. The most important and extensively studied transporter is the calcium transporter known as sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA) located in the membranes of ER/SR [88]. Few studies have revealed that autophagy and apoptosis are effectively triggered by SERCA inhibition in cancer cells; hence, SERCA is considered a novel therapeutic target for anticancer drugs [89, 90]. SERCA has a prominent role in tumor survival [91], and its inhibition causes a severe imbalance in calcium homeostasis in tumor cells, leading to activation of the ER stress response. This results in permanent damage to mitochondria by Ca2+ excess and affects the caspase and cytochrome-C release pathway [92].

Celastrol is reported to effectively mobilize the cytosolic calcium by directly suppressing ATP depletion and SERCA, thereby leading to autophagic and apoptotic cell death in MDR cancer cells. Autophagy is induced via the CaMKKβ-AMPK-mTOR signaling pathway. Additionally, celastrol effectively inhibits the ABC-transporter P-gp, which increases the sensitivity of MDR cancer cells and promotes the sensitization of cancer cells to taxol exposure [93]. These findings are in agreement with the findings of another study conducted by Liu et al., which reported that PERK phosphorylation and SERCA2B suppression are successfully modulated by celastrol, leading to autophagic cell death of cancer cells [94].

Celastrol induced EGFR degradation via autophagy regulation

Drug resistance is one of the alarming consequences of current anti-cancer therapies. In non-small cell lung cancer (NSCLC), the resistance is associated with a mutation in the epidermal growth factor receptor (EGFR). Celastrol shows selective cytotoxic activity against the EGFR mutant NSCLCs. Moreover, via the mechanism of calcium-mediated autophagy, celastrol significantly degrades EGFR and Akt expression in both mutant and wild type NSCLCs. Application of the autophagic inhibitor or calcium chelator blocks the degradation of EGFR and decreases cell death in H1975 gefitinib-resistant NSCLCs [95].

The relationship between autophagy, EGFR, and cancer has further been illustrated by So et al. [96]. This was verified by exposing NSCLCs to CK2 inhibitor; autophagy was induced, which subsequently downregulated the EGFR, leading to cell death. The autophagy triggered by CK2 inhibitors might differ from the autophagic pathways activated by celastrol, but it provides evidence that celastrol is a potential agent for the induction of autophagic-mediated EGFR degradation and can be an effective anticancer therapy for such resistant cancer cells [95].