Natural products extracted from distinct species significantly contributed to the development of effective therapeutics against all types of diseases. In this context, the ocean is of immense importance as it has a large reservoir of marine species with their biologically active compounds possessing various activities including anticancer, anti-inflammatory, antimicrobial, and antioxidant [36–39]. Several marine-derived secondary metabolites such as alkaloids, terpenes, peptides, and steroids exhibit potent anticancer activities [40, 41].
In the present study, we focused on heteronemin and evaluated its ferroptotic potential in a pancreatic cancer model. As previously reported, heteronemin exhibited anticancer, anti-nutritional, antimicrobial, protein inhibitory, and antitubercular activities [42, 43]. In agreement with our findings, heteronemin reduced cell viability and proliferation in several cancer cell lines including leukemia, colon adenocarcinoma, breast cancer, and renal carcinoma at a concentration of less than one micromolar [31, 44]. In the present study, heteronemin showed potent cytotoxic activity against Panc-1 cells with IC50 of 55 nM. We observed a good selectivity profile with an SI value of 4.65 for heteronemin in pancreatic cancer cells following 48 h of treatment.
The most promising strategies for PDAC treatment are to inhibit mutated genes, such as KRAS, to regulate macromolecules that contribute to the disease progression, or to overcome chemoresistance . The most used drugs approved by the FDA for pancreatic cancer are 5-fluorouracil, albumin-bound paclitaxel, cisplatin, gemcitabine, and FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, oxaliplatin) . These drugs have short half-lives and are usually given in higher and repeated doses which cause mild-to-moderate side effects [47–49]. Cisplatin is one of the agents used to treat pancreatic cancer. Severe side effects limit the therapeutic efficacy of cisplatin. Guo et al. (2018) reported that cisplatin inactivates GPX together with the induction of GSH depletion in cancer cells . Thus, we decided to use cisplatin as the positive control to compare its effect with heteronemin in PDAC. Similar to the short-term cell survival findings, the long-term cell survival results monitored in a colony formation assay supported the inhibitory effect of heteronemin, which was comparable to cisplatin. Heteronemin-treated cells showed a reduced migration ability as the concentration increased and the results were comparable to cisplatin. These observations indicated that heteronemin selectively inhibited cell growth and the results were comparable to the clinically used anticancer drugs in pancreatic cancer cells.
Chemotherapeutic agents disrupt cell homeostasis via inhibiting DNA synthesis, increasing oxidative stress, arresting the cell cycle, and inducing cellular death mechanisms such as necrosis and apoptosis. Although Bcl-mediated apoptotic pathway and autophagy were reported to be induced by heteronemin in cancer cells [29, 31], the effect of heteronemin on other cellular death pathways was not fully elucidated.
Recently, ferroptotic cell death is widely investigated in cancer studies [51, 52]. Most of the clinically used chemotherapeutic drugs were found to induce ferroptosis as well as apoptosis [51, 53]. Similarly, we observed that heteronemin failed to stimulate cell death in the presence of a ferroptosis inhibitor, Fer-1. In agreement with the current data, we reported that heteronemin induced cellular death can be rescued by ferroptosis inhibitor in hepatocellular carcinoma cells . These observations were critical for our further evaluation of heteronemin-induced ferroptosis in the present study. We hypothesized that heteronemin would regulate several pathways such as lipid peroxidation, iron transport, and iron storage to induce ferroptosis in cancer cells.
Increasing evidence demonstrated those numerous metabolic pathways contribute to ferroptosis through lipid-ROS production [54, 55]. Biochemical events including intracellular iron accumulation, and lipid peroxidation are critical for ferroptosis in cancer cells . Pathways inducing ferroptosis are associated with the reduction of cysteine uptake through the inhibition of system Xc- (SLC7A11), the reduction of GPX4 activity, and eventually the accumulation of intracellular lipid peroxides [57, 58]. GPX4 is a pivotal enzyme responsible for the detoxification of lipid peroxides and the progression of ferroptosis. Previously, the ability of heteronemin to reduce GPX4 protein expression was reported in hepatocellular carcinoma cell lines . In our study, GPX4 protein expression was significantly decreased in response to heteronemin as well. The downregulation of GPX4 by heteronemin together with the increased MDA levels indicated that heteronemin successfully inhibited the lipid peroxidation product scavenging activity of GPX4 and promoted ferroptosis in pancreatic cancer cells.
One of the components that distinguish ferroptosis from other cell death mechanisms is iron metabolism. Free Fe2+ causes ferroptosis by catalyzing free radical formation via Fenton reaction. Biochemically, the reduction of Fe3+ to Fe2+ is catalyzed by STEAP3 in the endosome. Fe2+ is released into the cytoplasm via DMT1 . Thus, any alteration in the expression of these proteins is critical for the labile iron pool and the consequent maintenance of iron homeostasis. Turcu et al. (2020) reported that blockade of DMT1 inhibits iron translocation which leads to lysosomal iron overload and ferroptosis in cancer stem cells . However, the upregulation of STEAP3 and DMT1 in pancreatic cancer cells following heteronemin treatment indicated that the conversion of Fe3+ to Fe2+ as well as the release of free Fe2+ into cytoplasm may be triggered by heteronemin.
Ferritinophagy is defined as the degradation of ferritin, providing free Fe2+ for the cell, and contributing to ferroptosis as a source of unstable iron ions [10, 59]. Previously, Atg5 and Atg7 knockdown/knockout were demonstrated to block erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation . Conversely, upregulation of Atg5 and Atg7 protein expressions in response to heteronemin treatment with slightly decreased ferritin light chain (FTL) protein level and lipid peroxidation may promote the induction of ferroptosis in cancer cells. Free iron accumulation inside the cell participated in the Fenton reaction to produce lipid peroxides, which was confirmed with the increased MDA levels.
Yang and Stockwell (2008) reported that cancer cells undergoing ferroptosis increased iron import and decreased iron storage when compared to other cells . Thus, it can be suggested that heteronemin sensitizes tumor cells to ferroptosis by modulating iron metabolism. Reduced iron storage because of decreased FTL and increased autophagy-related protein expression in response to heteronemin may contribute to iron overload and eventually trigger ferroptosis in cancer cells.