Therapeutic potential of thymoquinone in combination therapy against cancer and cancer stem cells

Table 1 Mode of action of the chemotherapeutic agents and cellular and molecular mechanism of action of the combination treatment in preclinical and clinical studies

Chemotherapeutic agent	Mode of action	Patients or animal model or cell lines	Cellular and molecular mechanism of action of the combination treatment	Ref.
Cyclophosphamide	Alkylates guanine base and causes the formation of DNA crosslinks leading to cell death	SKBR-3 and MDA-231 breast cancer cells	Increases the percentage of cells in G1 and sub- G1 phases. Downregulates the phosphorylation of Akt and the expression of cyclin D1 and upregulates PTEN	Emadi et al[25], Khan et al[27]
Temozolomide	Methylates DNA at specific sites on guanine and adenine bases causing cell demise	U87MG human glioblastoma multiforme cells	Increases the mitochondrial membrane potential disruption, cytochrome c release, ROS generation, DNA fragmentation and Bax/Bcl-2 ratio. Activates p53, caspases 9 and 3 and reduces NO and GSH levels. Reduces the expression and secretion of MMP-2 and MMP-9. Downregulates beclin-1 and ATG-7	Stupp et al[29], Khazaei et al[32], Pazhouhi et al[33], Pazhouhi et al[35]
Cisplatin	Interacts with purine bases and forms DNA crosslinks resulting in cell death	ID8-NGL mouse ovarian cancer cells. OVCAR3 and NCI/ADR-RES human ovarian cancer cells. BL/6 mice injected with ID8-NGL cells	Increases the level of Bax, pH2AX (ser139), cleaved caspase 3 and PARP. Decreases the level of PCNA and Ki67	Siddik et al[36], Wilson et al[39]
		Eca-109 human esophageal cancer cells. BALB/c nude mice inoculated with Eca-109 cells	Decreases the expression of p-STAT3, p-JAK2, Bcl-2, survivin and cyclin D1. Increases the expression of Bax and activates caspases 3, 7 and 9. Induces chromatin condensation and nuclear fragmentation	Hu et al[40]
		NCI-H460 non-small lung cancer cells. SCID mice injected with NCI-H460 cancer cells	Reduces the ratio of phosphor-Ser529 NFkB/NFkB	Jafri et al[42]
		UMSCC-14C head and neck squamous cancer cells and normal oral epithelial cells	Increases p53 and caspase 9 expression. Decreases Bcl-2 expression	Alaufi et al[43]
		SGC-7901 human gastric cancer cells. BALB/c mice implanted with gastric cancer cells	Increases the level of Bax, AIF, cytochrome c, cleaved caspases 9 and 3. Decreases the level of cyclin D1, Bcl-2, procaspases 9 and 3. Inhibits PI3K/Akt signaling pathway and downregulates P-gp by upregulating PTEN	Ma et al[44]
5-Fluorouracil	A pyrimidine analogue inhibiting the activity of thymidylate synthase enzyme causing the disruption of DNA synthesis and cell death	BGC-823, SGC-7901, MGC-803 and HGC-27 human gastric cancer cells. BALB/c athymic nude mice inoculated with gastric cancer cells	Increases the release of mitochondrial cytochrome c and the level of Bax, caspases 3 and 9. Decreases the level of Bcl-2 and induces nuclear fragmentation and chromatin condensation	Wilson et al[45], Lei et al[48]
		Azoxymethane-induced colorectal tumors in Wistar rats	Increases the expression of DKK-1, CDNK-1A, TGF-β1, TGF-βRII, Smad4 and GPx. Decreases the expression of Wnt, β-catenin, NFκB, COX-2, iNOS, VEGF and TBRAS	Kensara et al[49]
		HCT116, HT29 and SW620 human colon cancer cellsSW837 rectal cancer cells. Normal human intestinal epithelial cells. CAM tumors derived from HCT116 cells	Downregulates Wnt/β-catenin and PI3K/Akt pathways	Ndreshkjana et al[50]
		FADU nasopharyngeal cancer cells	Decreases the level of GSH	Williams et al[51]
		MG63 human osteosarcoma cells		Sarman et al[52]
Gemcitabine	A deoxycytidine analog preventing chain elongation during DNA synthesis causing cell death	PANC-1 and MIA PaCa-2 human pancreatic cancer cells	Downregulates PKM2 and decreases the expression of procaspase 3 and PARP	Moysan et al[53], Pandita et al[56]
		PANC-1, BxPC-3, and AsPC-1 human pancreatic cancer cell lines. BALB/c nude mice injected with PANC-1 cells	Downregulates Notch1, NICD, Bcl-2, Bcl-xL and XIAP. Inactivates Akt/mTOR/S6 signaling pathway and decreases the phosphorylation and nuclear translocation of p65. Upregulates PTEN, caspases 3 and 9 and Bax and increases cytochrome c release	Mu et al[57]
		MCF-7 and T47D human breast cancer cells	Increases pre-G1 cell population	Bashmail et al[58]
Paclitaxel	Inhibits microtubules disassembly and induces mitotic arrest	4T1 mouse breast cancer cells. Ehrlich tumor cells. Balb/c mice injected with Ehrlich tumor ascites cells	Increases the level of full length and cleaved caspases 3, 7 and 12 and PARP. Reduces phosphorylated p65 and Akt1. Modulates genes involved in apoptosis, cytokine -cytokine receptor interaction, Fas signaling, p53 signaling and JAK/STAT signaling	Ojima et al[59], Şakalar et al[63]
		MCF-7 and T47D human breast cancer cells	Increases pre-G1 cell population. Increases the level of cleaved caspase 3 and PARP and the expression of beclin-1 and LC3-II	Bashmail et al[64]
		MCF-7 human breast cancer cells		Soni et al[65]
Docetaxel	Inhibits microtubules disassembly and induces mitotic arrest	DU-145 human prostate cancer cells	Blocks PI3K/Akt signaling pathway and induces DNA fragmentation	Ojima et al[59], Dirican et al[69]
		DU-145 and C4-2B human prostate cancer cells	Inhibits PI3K/Akt signaling pathway. Increases the expression of Bax, Bid, caspase 3 and PARP and decreases the expression of Bcl-xL	Singh et al[70]
		MCF-7 and MDA-MB-231 human breast cancer cells	Induces DNA damage, cells shrinkage, nuclear fragments, apoptotic bodies and cytoplasmic vacuolation	Alkhatib et al[71]
		MCF-7 and MDA-MB-231 human breast cancer cells		Zafar et al[72]
		MCF-7 and MDA-MB-231 human breast cancer cells. Balb/c mice healthy or injected with Ehrlich ascites carcinoma cells	Induces nuclear fragmentation and restores the levels of oxidative stress parameters MDA, SOD and GSH. Prevents the alteration of blood cell count and serum biochemical parameters AST, ALT, creatinine and BUN	Zafar et al[73]
		MCF-7 breast cancer cells		Odeh et al[74]
Cabazitaxel	Inhibits microtubules disassembly and induces mitotic arrest	MCF-7 and MDA-MB-231 human breast cancer cells	Induces DNA fragmentation and increases the sub-G1 population	Ojima et al[59], Kommineni et al[78]
Doxorubicin	Intercalates DNA, inhibits topoisomerase II, forms free radicals when reduced leading to cell cycle arrest and cell death	Human HTLV-1 positive (HuT-102) and HTLV-1 negative (Jurkat) CD4+ malignant T-cell lines. NOD/SCID mice inoculated with HuT-102 tumor cells	Increases the sub-G1 population and induces ROS production. Disrupts the mitochondrial membrane potential. Downregulates the expression of NFkΒ and Ki67 and increases the phosphorylation of p53	Meredith et al[79], Fatfat et al[83]
		HL-60 acute myeloid leukemia cells. Dox resistant HT-29 colon carcinoma cells. MCF-7/TOPO multi-drug resistant breast cancer cells	Induces caspases 3 and 8 activity and ROS generation. Disrupts the mitochondrial membrane potential	Effenberger-Neidnicht et al[84]
		BALB/c OlaHsd-foxn1 nude mice injected with MDA-MB-231 breast cancer cells	Induces p38 MAPK phosphorylation and inhibit the expression of XIAP, survivin, Bcl-xL and Bcl-2	Woo et al[85]
		SMMC-7721 and HepG2 hepatocarcinoma cells and human normal liver cells HL-7702	Increases caspase 3 and PARP cleavage	Jehan et al[86]
		MDA-MB-231 human breast cancer cells. MCF-10A and 3T3 non-neoplastic cells	Induces cell shrinkage, membrane blebbing and apoptotic bodies and disrupts the cell membrane. Increases the Sub-G0 population	Ibiyeye et al[87]
		MCF-7 human breast adenocarcinoma and HEPG2 human hepatocellular carcinoma. Albino mice implanted with Heps murine liver cancer cells	Decreases NFkB level and increases that of caspase 3. Increases the level of renal antioxidant enzymes SOD and catalase. Modulates the level of renal oxidative stress biomarkers GSH and MDA. Decreases the level of nephrotoxicity biomarkers BUN and serum creatinine	Zidan et al[88]
		Albino transplanted with Ehrlich carcinoma cells	Upregulates p53 and reduces the level of Bcl-2. Decreases the level of cardiac MDA. Decreases the serum level of cardiac markers lactate and creatine	El-Ashmawy et al[89]
Topotecan	Inhibits DNA topoisomerase I and causes the formation of irreversible DNA double stranded breaks resulting in cell death. Inhibits hypoxia-inducible factor 1α	U937 acute myelogenous leukemia cells	Increases the sub-G1 population. Increases the expression level of Bax/Bcl-2, p53 and p21 and the cleavage of caspases 3 and 9	Robati et al[90], Khalife et al[95]
		HT-29 human colon cancer cells	Increases the sub-G1 population. Has no effect on p53, Bax and Bcl-2 expression	Khalife et al[96]
Bortezomib	Inhibits the proteasome	U266, H929, KMS, RPMI-8226, RPMI-8226-Dox-6 (doxorubicin-resistant clone), RPMI-8226-LR-5 (a melphalan-resistant clone) human multiple myeloma cells. Balb/c mice implanted with U266 cells	Increases the sub-G1 population and the cleavage of caspase 3 and PARP. Reduces the phosphorylation of NFkB (p65) and the expression of Ki67, VEGF, Bcl-2 and the serum levels of IL-6 and TNF-α	Siveen et al[99]
Imatinib	Inhibits tyrosine kinase	HCT116 human colorectal cancer cells	Decreases the expression of ABCB1, ABCG2 and hOCT1. Increases the uptake/efflux ratio of imatinib	Thabet et al[103]
Tamoxifen	Competes with estrogen and estradiol for the binding to their receptors and modulates their signaling pathway	MCF-7 and MDA-MB-231 human breast cancer cells		Day et al[104], Ganji-Harsini et al[106]
		MCF-7, MDA-MB-231, MDA-MB-468, T47D, NIH/3T3 and HaCaT human breast cancer cells. Athymic BALB/c mice injected with MDA-MB-231 cells	Decreases the expression of XIAP and the level of p-Akt, p-Bad, p-MAPK and p-GSK-3β and downregulates the expression of Bcl-xL, Bcl-2 and Ki67. Increases the cleavage of caspase 9 and PARP and induces the expression of Bax, AIF, cytochrome c and p27. Increases the percentage of cells in sub-G1 phase and the fragmentation of DNA	Rajput et al[107]
		Breast cancer patients	Increases the tumor tissue catalase, SOD and caspase 3. Decreases the tumor tissue Bcl-2, TGF-β1, MDA, TNF-α and IL-6	Kabel et al[108]
Zoledronic acid	Inhibits osteoclast-mediated bone resorption	PC-3 and DU- 145 human prostate cancer cells	Increases DNA fragmentation and activates caspases 3 and 7	Polascik et al[109], Dirican et al[112]
Arsenic trioxide		Human HTLV-I positive (HuT-102 and C91) and HTLV-I negative (CEM and Jurkat) malignant T-cell lines. NOD SCID mice inoculated with HuT-102 cells	Increases the percentage of cells in Pre-G1 phase, the disruption of the mitochondrial membrane potential and the cleavage of PARP and caspase 3. Upregulates p53, Bax and downregulates XIAP and Bcl- 2	Houssein et al[117]

PTEN: Phosphatase and tensin homolog; ROS: Reactive oxygen species; Bax: Bcl-2-associated X protein; NO: Nitric oxide; GSH: Glutathione; MMP: Matrix metalloproteinase; ATG-7: Autophagy-related 7; pH2AX: Phospho-histone 2AX; PCNA: Proliferating cell nuclear antigen; JAK2: Janus kinase 2; STAT3: Signal transducer and activator of transcription 3; NFkB: Nuclear factor kappa B; PI3K: Phosphatidylinositol-3-kinase; AIF: Apoptosis inducing factor; PARP: Poly (ADP-ribose) polymerases; CAM: Chorioallantoic membrane; MAPK: Mitogen-activated protein kinase; COX-2: Cyclooxygenase 2; iNOS: Inducible nitric oxide synthase; VEGF: Vascular endothelial growth factor; TBRAS: Thiobarbituric acid reactive substances; DKK-1: Dickkopf-related protein-1; CDNK-1A: Cyclin-dependent kinase inhibitor 1A; TGF-β1: Tak transforming growth factor beta 1; TGF-βRII: Transforming growth factor, beta receptor II; GPx: Glutathione peroxidase; GSH: Glutathione; XIAP: X-linked inhibitor of apoptosis protein; mTOR: Mammalian target of rapamycin; PKM2: Pyruvate kinase M2; Bid: BH3 interacting-domain death agonist; AST: Aspartate transaminase; ALT: Alanine transaminase; MDA: Malondialdehyde; SOD: Superoxide dismutase; BUN: Blood urea nitrogen; IL-6: Interleukin 6; TNF-α: Tumor necrosis factor alpha; GSK-3β: Glycogen synthase kinase 3 beta; ABCB1A: ATP-binding cassette subfamily B member 1; ABCG2: ATP-binding cassette subfamily G member 2; hOCT1: Human organic cation transporter 1.

Table 2 Cellular and molecular mechanism of action of the combination treatment in preclinical studies

Therapeutic agent	Animal model or cell line	Cellular and molecular mechanism of action of the combination treatment	Ref.
Radiation	MCF-7 and T47D human breast cancer cells	Increases the percentage of cells in sub-G1 phase	Velho-Pereira et al[121]
	MCF-7 and MDA-MB-231 human breast cancer cells	Restores the expression levels of TGF-β and its downstream molecules NFkB, Smad2, Snail and Twist, adhesion molecules E-cadherin and cytokeratin 19, mesenchymal markers integrin αV, MMP-9, and MMP-2	Rajput et al[122]
	B16-F10 melanoma cells	Inhibits the phosphorylation of JAK2 and STAT3. Increases the expression of caspase 3 and Bax. Reduce the expression of Bcl-2 and survivin and the level of VEGF-A, MCP-1, TGF-β1, RANTES and IL-1β. Induces DNA damage	Hatiboglu et al[123]
microRNA-34a	BT-549 metastatic breast cancer cells	Targets and downregulates TWIST1 and ZEB1	Imani et al[126]
Akt-siRNA	Akt-overexpressing MCF-7 and T47D. Tamoxifen resistant MCF-7 and T47D breast cancer cells. BALB/c mice injected with MCF-7/TAM cells	Reduces Akt expression and MDM-2 activation. Activates p53, increases the level of Bax and Bim and decreases the level of Bcl-2 and Ki67	Rajput et al[127]
Vitamin D3	Azoxymethane-induced colorectal tumors in Wistar rats	Reduces the level of Wnt, β-catenin, NFkB, COX-2, iNOS, VEGF and HSP-90 and increases that of DKK-1, CDNK-1A, TGF-β1, TGF-β/RII and Smad4	Mohamed et al[131]
Melatonin	EMT6/P mouse breast cancer cells. Balb/C mice transplanted with EMT6/P cells	Reduces the expression of VEGF and the serum level of AST and ALT. Increases the serum level of IFN-α and decreases that of IL-4	Odeh et al[134]
Artemisinin	CCRF-CEM and multidrug-resistant CEM/ADR5000 human leukemia cells. Healthy human foreskin fibroblasts		Fröhlich et al[136]
Artesunic acid	HCT116, HT29, Caco-2, DLD-1 colon cancer cells. HCEC nonmalignant colon epithelial cells	Induces ROS generation, DNA damage, PARP and caspase 9 cleavage. Increases the level of ɣ-H2AX	Fröhlich et al[137]
Diosgenin	A431 and Hep2 human squamous cell carcinoma. Swiss albino mice injected with sarcoma 180 cells	Induces DNA fragmentation and cytoskeletal changes. Decreases the expression of CD31 and Ki67	Das et al[138]
Emodin	MCF-7, MDA-MB-231, MDA-MB-468 and T47D human breast cancer cells. CAM inoculated with MCF-7 cells	Increases the percentage of cells in sub-G1 phase. Increases ROS generation, cytochrome c release, expression levels of p53, Bax and cleaved caspase 3. Reduces Bcl-2, pFAK and integrinβ1 expression level. Induces nuclear fragmentation, shrinkage, apoptotic body formation, chromatin condensation and membrane blebbing	Bhattacharjee et al[140]
Ferulic acid	MDA-MB-231 human breast cancer cells		Al-Mutairi et al[143]
Genistein	CALC-62 and ACC448 human thyroid cells derived from anaplastic carcinoma CGTH-W1, ACC360 derived from follicular carcinoma	Reduces the expression level of human telomerase reverse transcriptase, VEGF-A and NFkB. Increases the expression level of PTEN and p21 and activates caspase 3	Ozturk et al[145]
Indirubin-3-monoxime	A549 human lung cancer cells. HFL-1 human fetal lung fibroblast. CD1-nude mice injected with A549 cells	Increases the percentage of cells in Sub-G0 phase. Reduces Bcl-2/Bax ratio, TNF-α release and p-Akt (s473), p-mTOR, NFkB/p65, caspase3 and p53 expression level	Dera et al[147]
Piperine	EMT6/P mouse mammary cancer cells. Balb/C female mice injected with EMT6/P cancer cells	Reduces VEGF expression. Increases IFN-γ and IL-2 level and caspase 3 activity	Talib et al[149]
	HepG2 human hepatocellular cancer cells	Increase ROS generation and decreases GSH and NADPH level	Das et al[151]
Resveratrol	HepG2 human hepatocellular cancer cells	Increases caspase 3 activity. Decreases GSH and MDA level	Ismail et al[153]
	EMT6/p mouse epithelial breast cancer cells. MCF-7 and T47D human epithelial breast cancer cells kidney epithelial cells. Balb/C mice injected with EMT6/p cancer cells	Induces DNA fragmentation and increases IFN-γ and IL-4 level. Reduces VEGF expression	Alobaedi et al[154]
Selenium	MG-63 human osteosarcoma cell line	Increases cellular damage, and decreases the level of alkaline phosphatase and GSH	Barron et al[156]

PTEN: Phosphatase and tensin homolog; ROS: Reactive oxygen species; Bax: Bcl-2-associated X protein; GSH: Glutathione; MMP: Matrix metalloproteinases; ɣ-H2AX: Gamma-histone 2AX; JAK2: Janus kinase 2; STAT3: Signal transducer and activator of transcription 3; NFkB: Nuclear factor kappa B; COX-2: Cyclooxygenase 2; iNOS: Inducible nitric oxide synthase; VEGF: Vascular endothelial growth factor; DKK-1: Dickkopf-related protein-1; CDNK-1A: Cyclin-dependent kinase inhibitor 1A; TGF-β1: Transforming growth factor beta 1; TGF-βRII: Transforming growth factor beta receptor II; GSH: Glutathione; mTOR: Mammalian target of rapamycin; AST: Aspartate transaminase; ALT: Alanine transaminase; MDA: Malondialdehyde; IL: Interleukin; INF: Interferon; TNF-α: Tumor necrosis factor alpha; MCP-1: Monocyte chemoattractant protein-1; RANTES: Regulated on activation normal T cell expressed sequence; TWIST1: Twist-related protein 1; ZEB1: Zinc finger E-box binding homeobox 1; MDM-2: Mouse double minute 2; NADPH: Nicotinamide-adenine dinucleotide phosphate; CAM: Chorioallantoic membrane.