Targeting Triple-Negative Breast Cancer by the Phytopolyphenol Carnosol: ROS-Dependent Mechanisms.

Triple-negative breast cancer (TNBC), which lacks the expression of the three hormone receptors (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor), is characterized by a high proliferative index, high invasiveness, poor prognosis, early relapse, and a tendency to be present in advanced stages. These characteristics rank TNBC among the most aggressive and lethal forms of breast cancer. The lack of the three receptors renders conventional hormonal therapy ineffective against TNBC. Moreover, there are no clinically approved therapies that specifically target TNBC, and the currently used chemotherapeutic agents, such as cisplatin, taxanes, and other platinum compounds, have a limited clinical effect and develop chemoresistance over time. Phytochemicals have shown efficacy against several types of cancer, including TNBC, by targeting several pathways involved in cancer development and progression. In this review, we focus on one phytochemical carnosol, a natural polyphenolic terpenoid with strong anti-TNBC effects and its ROS-dependent molecular mechanisms of action. We discuss how carnosol targets key pathways and proteins regulating the cell cycle, growth, epigenetic regulators, invasion, and metastasis of TNBC. This review identifies carnosol as a potential novel targeting protein degradation molecule.

Corrigendum: Carnosol, a natural polyphenol, inhibits migration, metastasis, and tumor growth of breast cancer via a ROS-dependent proteasome degradation of STAT3.

[This corrects the article DOI: 10.3389/fonc.2019.00743.].

Carnosol Induces p38-Mediated ER Stress Response and Autophagy in Human Breast Cancer Cells.

We recently reported that carnosol induces ROS-dependent autophagy and apoptosis in breast cancer cells. We also reported that carnosol inhibits breast cancer cell migration, invasion, and in ovo tumor growth, as well as targets STAT3, PCAF, and p300 to proteasome degradation. Here, we investigated the molecular mechanisms underlying its anti-malignant activity in breast cancer. We report that carnosol induces a ROS-dependent type I and type II programmed cell death (PCD-I or PCD-II, respectively), which occurred independently of each other. Indeed, chemical inhibition of autophagy had no effect on the induction of apoptosis, evident by the absence of cleaved PARP. Electron microscopy revealed that carnosol-treated cells exhibited enlarged endoplasmic reticulum, characteristic of ER stress. Markers of the three unfolded protein response pathways (PERK, IRE-1 α, and ATF6), namely ATF4, CHOP, phospho-IRE-1α, XBP1S, and cleaved ATF6 were upregulated in a ROS-dependent manner. In addition, carnosol induced a ROS-dependent activation of p38MAPK, increased the overall level of protein polyubiquitination, and targeted mTOR protein to proteasome degradation. Interestingly, inhibition of p38MAPK, by SB202190 and 203580, reduced cell death, selectively blocked the induction of IRE-1α and ATF6 UPR sensors and inhibited autophagy. In addition, inhibition of p38 reduced the carnosol-induced polyubiquitination and rescued mTOR, PCAF, and STAT3 from proteasomal degradation. Importantly, activation of PERK sensors and induction of apoptosis occurred independently of p38 activation. Taken together, our results suggest that ROS-dependent induced-ER stress contributes to carnosol-induced apoptotic and autophagic cell death in breast cancer cells, and further confirm that carnosol is a promising agent for breast cancer therapy.

Carnosol Is a Novel Inhibitor of p300 Acetyltransferase in Breast Cancer.

Carnosol, a natural polyphenol abundant in edible plants such as sage, rosemary, and oregano, has shown promising anticancer activity against various types of cancers. Nonetheless, very little is known about its molecular mechanism of action or its downstream target(s). We have previously shown that carnosol inhibits cellular proliferation, migration, invasion, and metastasis as well as triggers autophagy and apoptosis in the highly invasive MDA-MB-231 breast cancer cells. Here, we report that carnosol induces histone hypoacetylation in MDA-MB-231 and Hs578T breast cancer cells. We show that, while carnosol does not affect HDACs, it promotes a ROS-dependent proteasome degradation of p300 and PCAF histone acetyl transferases (HATs) without affecting other HATs such as GCN5 and hMOF. Carnosol-induced histone hypoacetylation remains persistent even when p300 and PCAF protein levels were rescued from degradation by (i) the inhibition of the proteasome activity by the proteasome inhibitors MG-132 and bortezomib, and (ii) the inhibition of ROS accumulation by the ROS scavenger, N-acetylcysteine. In addition, we report that, in a cell-free system, carnosol efficiently inhibits histone acetyltransferase activity of recombinant p300 but not that of PCAF or GCN5. Molecular docking studies reveal that carnosol inhibits p300 HAT activity by blocking the entry of the acetyl-CoA binding pocket of the catalytic domain. The superimposition of the docked conformation of the p300 HAT domain in complex with carnosol shows a similar orientation as the p300 structure with acetyl-CoA. Carnosol occupies the region where the pantetheine arm of the acetyl-CoA is bound. This study further confirms carnosol as a promising anti-breast cancer therapeutic compound and identifies it as a novel natural p300 inhibitor that could be added to the existing panel of inhibitors.

Pharmacological and Antioxidant Activities of Rhus coriaria L. (Sumac).

Rhus coriaria L. (Anacardiaceae), commonly known as sumac, is a commonly used spice, condiment, and flavoring agent, especially in the Mediterranean region. Owing to its bountiful beneficial values, sumac has been used in traditional medicine for the management and treatment of many ailments including hemorrhoids, wound healing, diarrhea, ulcer, and eye inflammation. This plant is rich in various classes of phytochemicals including flavonoids, tannins, polyphenolic compounds, organic acids, and many others. By virtue of its bioactive, Rhus coriaria possesses powerful antioxidant capacities that have ameliorative and therapeutic benefits for many common diseases including cardiovascular disease, diabetes, and cancer. This review describes the phytochemical properties of R. coriaria and then focuses on the potent antioxidant capacities of sumac. We then dissect the cellular and molecular mechanisms of sumac's action in modulating many pathophysiological instigators. We show how accumulating evidence supports the antibacterial, antinociceptive, antidiabetic, cardioprotective, neuroprotective, and anticancer effects of this plant, especially that toxicity studies show that sumac is very safe to consume by humans and has little toxicity. Taken together, the findings we summarize here support the utilization of this plant as an attractive target for drug discovery.

Origanum majorana Essential Oil Triggers p38 MAPK-Mediated Protective Autophagy, Apoptosis, and Caspase-Dependent Cleavage of P70S6K in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common type of cancer in terms of incidence and mortality worldwide. Here we have investigated the anti-colon cancer potential of Origanum majorana essential oil (OMEO) and its underlying mechanisms of action. We showed that OMEO significantly inhibited the cellular viability and colony growth of human HT-29 colorectal cancer cells. OMEO induced protective autophagy, associated with downregulation of the mTOR/p70S6K pathway, and activated caspase-8 and caspase-9-dependent apoptosis. Blockade of autophagy with 3-methyladenine (3-MA) and chloroquine (CQ), two autophagy inhibitors, potentiated the OMEO-induced apoptotic cell death. Inversely, inhibition of apoptosis with the pan-caspase inhibitor, Z-VAD-FMK, significantly reduced cell death, suggesting that apoptosis represents the main mechanism of OMEO-induced cell death. Mechanistically, we found that OMEO induces protective autophagy and apoptotic cells death via the activation of the p38 MAPK signaling pathway. Pharmacological inhibition of p38 MAPK by the p38 inhibitors SB 202190 and SB 203580 not only significantly decreased apoptotic cell death, but also reduced the autophagy level in OMEO treated HT-29 cells. Strikingly, we found that OMEO also induces p38 MAPK-mediated caspase-dependent cleavage of p70S6K, a protein reported to be overexpressed in colon cancer and associated with drug resistance. Our findings suggest that OMEO inhibits colon cancer through p38 MAPK-mediated protective autophagy and apoptosis associated with caspase-dependent cleavage of p70S6K. To the best of our knowledge, this study is the first to report on the implications of the p38 MAPK signaling pathway in targeting p70S6K to caspase cleavage.

Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway.

Colorectal cancer is considered as the third leading cause of cancer death. In the present study, we investigated the potential anticancer effect and the molecular mechanism of Origanum majorana ethanolic extract (OME) against human colorectal cancer cells. We showed that OME exhibited strong anti-proliferative activity in a concentration- and time-dependent manner against two human colorectal cancer cell lines (HT-29 and Caco-2). OME inhibited cell viability, colony growth and induced mitotic arrest of HT-29 cells. Also, OME induced DNA damage, triggered abortive autophagy and activated a caspase 3 and 7-dependent extrinsic apoptotic pathway, most likely through activation of the TNFα pathway. Time-course analysis revealed that DNA damage occurred concomitantly with abortive autophagy after 4 h post-OME treatment while apoptosis was activated only 24 h later. Blockade of autophagy initiation, by 3-methyladenine, partially rescued OME-induced cell death. Cell viability arose from 37% in control group to 67% in group pre-treated with 3-MA before addition of OME. Inhibition of apoptosis, however, had a minimal effect on cell viability; it rose from 37% in control group to 43% in group pre-treated with Z-VAD-FMK. We also found that OME downregulated survivin in HT-29 cells. Our findings provide a strong evidence that O. majorana extract possesses strong anti-colon cancer potential, at least, through induction of autophagy and apoptosis. These finding provide the basis for therapeutic potential of O. majorana in the treatment of colon cancer.

Carnosol, a Natural Polyphenol, Inhibits Migration, Metastasis, and Tumor Growth of Breast Cancer via a ROS-Dependent Proteasome Degradation of STAT3.

We have previously demonstrated that carnosol, a naturally occurring diterpene, inhibited in vitro cell viability and colony growth, as well as induced cell cycle arrest, autophagy and apoptosis in human triple negative breast cancer (TNBC) cells. In the present study, we evaluated the ability of carnosol to inhibit tumor growth and metastasis in vivo. We found that non-cytotoxic concentrations of carnosol inhibited the migration and invasion of MDA-MB-231 cells in wound healing and matrigel invasion assays. Furthermore, gelatin zymography, ELISA, and RT-PCR assays revealed that carnosol inhibited the activity and downregulation the expression of MMP-9. Mechanistically, we demonstrated that carnosol suppressed the activation of STAT3 signaling pathway through a ROS-dependent targeting of STAT3 to proteasome-degradation in breast cancer cells (MDA-MB-231, Hs578T, MCF-7, and T47D). We show that blockade of proteasome activity, by MG-132 and bortezomib, or ROS accumulation, by N-acetylcysteine (NAC), restored the level of STAT3 protein. In addition, using chick embryo tumor growth assay, we showed that carnosol significantly and markedly suppressed tumor growth and metastasis of breast cancer xenografts. To the best of our knowledge, this is the first report which shows that carnosol specifically targets signal transducer and activator of transcription 3 (STAT3) for proteasome degradation in breast cancer. Our study further provide evidence that carnosol may represent a promising therapeutic candidate that canmodulate breast cancer growth and metastasis.