Mitochondrial calcium overload contributes to cannabinoid-induced paraptosis in hormone-responsive breast cancer cells

Abstract

Studies have shown that natural products can induce paraptosis in tumour cell lines. Paraptosis is characterized by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and mitochondria. The mechanism of paraptosis is unclear; however, dysregulation of Ca²⁺ homeostasis is believed to affect paraptosis induction. This study investigated the mechanism of cell death induced by a phytocannabinoid ratio in the MCF7 breast cancer cell line. The crystal violet assay was used to detect changes in viability and morphology changes were investigated using light and transmission electron microscopy. Various inhibitors, fluorescent staining with high-content screening, and Western blot analysis were used to investigate different cell death mechanisms. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, no apoptosis, necrosis, autophagy, or ferroptosis was detected. Vacuolation induced by phytocannabinoid treatment was inhibited by cycloheximide, suggesting paraptosis induction. The mechanism of paraptosis induction was investigated, and it was found that treatment (1) induced ER dilation and mitochondrial swelling, (2) induced significant ER stress and mitochondrial Ca²⁺ overload and dysfunction, which appeared to be mediated by the voltage-dependent anion channel, and (3) significantly impaired all mitochondrial metabolic pathways. The data demonstrated that paraptosis induced by the cannabinoid ratio was mediated by Ca²⁺ flux from the ER to the mitochondria. These findings highlight a novel mechanism of cannabinoid-induced cell death and emphasize the anti-cancer potential of cannabinoid ratios, which exhibited enhanced effects compared to individual cannabinoids.

References

[1] SungH, FerlayJ, SiegelRL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209-249.

[2] GiaquintoAN, SungH, MillerKD, et al. Breast cancer statistics, 2022. CA Cancer J Clin. 2022;72:524-541.

[3] McAndrewNP, FinnRS. Clinical review on the management of hormone receptor–positive metastatic breast cancer. JCO Oncol Pract. 2022;18:319-327.

[4] FerreiraAR, di Meglio A, PistilliB, et al. Differential impact of endocrine therapy and chemotherapy on quality of life of breast cancer survivors: a prospective patient-reported outcomes analysis. Ann Oncol. 2019;30:1784-1795.

[5] MustafaG, ArifR, AttaA, Sharif S, JamilA. Bioactive compounds from medicinal plants and their importance in drug discovery in Pakistan. Matrix Sci Pharma. 2017;1:17-26.

[6] FordjourE, ManfulCF, SeyAA, et al. Cannabis: a multifaceted plant with endless potentials. Front Pharmacol. 2023;14:1-36.

[7] RussoEB. Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br J Pharmacol. 2011;163:1344-1364.

[8] DevaneWA, DysarzFA 3rd, JohnsonMR, Melvin LS, HowlettAC. Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol. 1988;34:605-613.

[9] MunroS, ThomasKL, Abu-ShaarM. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61-65.

[10] Di MarzoV. New approaches and challenges to targeting the endocannabinoid system. Nat Rev Drug Discov. 2018;17:623-639.

[11] Di MarzoV, De Petrocellis L. Why do cannabinoid receptors have more than one endogenous ligand? Philos Trans R Soc Lond B Biol Sci. 2012;367:3216-3228.

[12] MunsonAE, HarrisLS, FriedmanMA, Dewey WL, CarchmanRA. Antineoplastic activity of Cannabinoids. J Natl Cancer Inst. 1975;55:597-602.

[13] AlmeidaCF, Teixeira N, Correia-da-SilvaG, AmaralC. Cannabinoids in breast cancer: differential susceptibility according to subtype. Molecules. 2022;27:156.

[14] VelascoG, Sánchez C, GuzmánM. Anticancer mechanisms of cannabinoids. Curr Oncol. 2016;23:S23-S32.

[15] MangalN, Erridge S, HabibN, SadanandamA, ReebyeV, SodergrenMH. Cannabinoids in the landscape of cancer. J Cancer Res Clin Oncol. 2021;147:2507-2534.

[16] de la HarpeA, BeukesN, FrostCL. CBD activation of TRPV1 induces oxidative signaling and subsequent ER stress in breast cancer cell lines. Biotechnol Appl Biochem. 2022;69:420-430.

[17] BaramL, PeledE, BermanP, et al. The heterogeneity and complexity of cannabis extracts as antitumor agents. Oncotarget. 2019;10:4091-4106.

[18] SperandioS, de Belle I, BredesenDE. An alternative, nonapoptotic form of programmed cell death. Proc Natl Acad Sci. 2000;97:14376-14381.

[19] HansonS, DharanA, P VJ, et al. Paraptosis: a unique cell death mode for targeting cancer. Front Pharmacol. 2023;14:1-29.

[20] FontanaF, Raimondi M, MarzagalliM, Di DomizioA, Limonta P. The emerging role of paraptosis in tumor cell biology: perspectives for cancer prevention and therapy with natural compounds. Biochim Biophys Acta Rev Cancer. 2020;1873:188338.

[21] LeeD, KimIY, SahaS, Choi KS. Paraptosis in the anti-cancer arsenal of natural products. Pharmacol Ther. 2016;162:120-133.

[22] WasikAM, Almestrand S, WangX, et al. WIN55, 212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells. Cell Death Dis. 2011;2:e225.

[23] SchoemanR, BeukesN, FrostC. Cannabinoid combination induces cytoplasmic vacuolation in MCF-7 breast cancer cells. Molecules. 2020;25:4682.

[24] FrostCL, BeukesN, SchoemanRLS. Cannabinoid combinations and their use in the treatment of cancer. EP4203925 2023.

[25] FeoktistovaM, Geserick P, LeverkusM. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb Protoc. 2016;4:pdb.prot087379.

[26] RaimondiM, Fontana F, MarzagalliM, et al. Ca(2+) overload-and ROS-associated mitochondrial dysfunction contributes to δ-tocotrienol-mediated paraptosis in melanoma cells. Apoptosis. 2021;26:277-292.

[27] BradfordMM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.

[28] ZaiontzC. Real Statistics Using Excel. www.real-statistics.com 2023.

[29] Prasad Tharanga JayasooriyaRG, DilsharaMG, Molagoda IMN, et al. Camptothecin induces G(2)/M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: implications of reactive oxygen species. Oncotarget. 2018;9:21744-21757.

[30] WuYT, TanHL, ShuiG, et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem. 2010;285:10850-10861.

[31] ZhongJ-T, YuJ, WangHJ, et al. Effects of endoplasmic reticulum stress on the autophagy, apoptosis, and chemotherapy resistance of human breast cancer cells by regulating the PI3K/AKT/mTOR signaling pathway. Tumor Biol. 2017;39:1010428317697562.

[32] DixonSJ, Lemberg KM, LamprechtMR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149:1060-1072.

[33] YangWS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008;15:234-245.

[34] KimSH, ShinHY, KimYS, et al. Tunicamycin induces paraptosis potentiated by inhibition of BRAFV600E in FRO anaplastic thyroid carcinoma cells. Anticancer Res. 2014;34:4857-4868.

[35] LeeAS. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods. 2005;35:373-381.

[36] NishitohH. CHOP is a multifunctional transcription factor in the ER stress response. J Biochem. 2011;151:217-219.

[37] WangX, RonD. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase. Science. 1996;272:1347-1349.

[38] ZinsznerH, KurodaM, WangX, et al. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev. 1998;12:982-995.

[39] YoonMJ, KimEH, LimJH, Kwon TK, ChoiKS. Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells. Free Radic Biol Med. 2010;48:713-726.

[40] YoonMJ, KimEH, KwonTK, Park SA, ChoiKS. Simultaneous mitochondrial Ca2+ overload and proteasomal inhibition are responsible for the induction of paraptosis in malignant breast cancer cells. Cancer Lett. 2012;324:197-209.

[41] YoonMJ, LeeAR, JeongSA, et al. Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells. Oncotarget. 2014;5:6816-6831.

[42] LeeDM, KimIY, SeoMJ, Kwon MR, ChoiKS. Nutlin-3 enhances the bortezomib sensitivity of p53-defective cancer cells by inducing paraptosis. Exp Mol Med. 2017;49:e365.

[43] ParadiesG, Paradies V, RuggieroFM, PetrosilloG. Role of cardiolipin in mitochondrial function and dynamics in health and disease: molecular and pharmacological aspects. Cells. 2019;8(7):728.

[44] KimE, LeeDM, SeoMJ, Lee HJ, ChoiKS. Intracellular Ca2 + imbalance critically contributes to paraptosis. Front Cell Dev Biol. 2021;8:607844.

[45] KeinanN, Tyomkin D, Shoshan-BarmatzV. Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis. Mol Cell Biol. 2010;30:5698-5709.

[46] Shoshan-BarmatzV, Hadad N, FengW, et al. VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle. FEBS Lett. 1996;386:205-210.

[47] KosmachA, RomanB, SunJ, et al. Monitoring mitochondrial calcium and metabolism in the beating MCU-KO heart. Cell Rep. 2021;37:109846.

[48] KaasikA, Safiulina D, ZharkovskyA, VekslerV. Regulation of mitochondrial matrix volume. Am J Physiol Cell Physiol. 2007;292:C157-C163.

[49] JavadovS, Chapa-Dubocq X, MakarovV. Different approaches to modeling analysis of mitochondrial swelling. Mitochondrion. 2018;38:58-70.

[50] HunterDR, Haworth R, SouthardJ. Relationship between configuration, function, and permeability in calcium-treated mitochondria. J Biol Chem. 1976;251:5069-5077.

[51] Matuz-MaresD, González-Andrade M, Araiza-VillanuevaMG, Vilchis-LanderosMM, Vázquez-MezaH. Mitochondrial calcium: effects of its imbalance in disease. Antioxidants. 2022;11(5):801.

[52] WongR, Steenbergen C, MurphyE. Mitochondrial permeability transition pore and calcium handling. Methods Mol Biol. 2012;810:235-242.

[53] VandecaetsbeekI, Vangheluwe P, RaeymaekersL, WuytackF, Vanoevelen J. The Ca2+ pumps of the endoplasmic reticulum and Golgi apparatus. Cold Spring Harb Perspect Biol. 2011;3:a004184.

[54] MarksAR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol-Heart Circ Physiol. 1997;272:H597-H605.

[55] VanceJE. Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem. 1990;265:7248-7256.

[56] GincelD, ZaidH, Shoshan-BarmatzV. Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. Biochem J. 2001;358:147-155.

[57] RapizziE, PintonP, SzabadkaiG, et al. Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria. J Cell Biol. 2002;159:613-624.

[58] BaughmanJM, Perocchi F, GirgisHS, et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature. 2011;476:341-345.

[59] De StefaniD, Raffaello A, TeardoE, SzabòI, Rizzuto R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature. 2011;476:336-340.

[60] YumnamS, HongGE, RahaS, et al. Mitochondrial dysfunction and Ca2+ overload contributes to hesperidin induced paraptosis in hepatoblastoma cells, HepG2. J Cell Physiol. 2016;231:1261-1268.

[61] XueJ, LiR, ZhaoX, et al. Morusin induces paraptosis-like cell death through mitochondrial calcium overload and dysfunction in epithelial ovarian cancer. Chem Biol Interact. 2018;283:59-74.

[62] RimmermanN, Ben-Hail D, PoratZ, et al. Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death. Cell Death Dis. 2013;4:e949.

[63] Olivas-AguirreM, Torres-López L, Valle-ReyesJS, Hernández-CruzA, Pottosin I, DobrovinskayaO. Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia. Cell Death Dis. 2019;10:779.

[64] TanW, Colombini M. VDAC closure increases calcium ion flux. Biochim Biophys Acta (BBA) Biomembranes. 2007;1768:2510-2515.

[65] HanahanD, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646-674.

[66] MoranaO, WoodW, GregoryCD. The apoptosis paradox in cancer. Int J Mol Sci. 2022;23:1328.

[67] BabickiS, ArndtD, MarcuA, et al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res. 2016;44:W147-W153.

[68] RyuW-I, CohenBM, SonntagK-C. Hypothesis and theory: characterizing abnormalities of energy metabolism using a cellular platform as a personalized medicine approach for Alzheimer’s disease. Front Cell Dev Biol. 2021;9:697578.

[69] ZhaoY, ButlerEB, TanM. Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis. 2013;4:e532.

[70] Méndez-LucasA, Lin W, DriscollPC, et al. Identifying strategies to target the metabolic flexibility of tumours. Nat Metab. 2020;2:335-350.

[71] StineZE, SchugZT, SalvinoJM, Dang CV. Targeting cancer metabolism in the era of precision oncology. Nat Rev Drug Discov. 2022;21:141-162.

RIGHTS & PERMISSIONS

2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.