Thymoquinone enhances the antioxidant and anticancer activity of Lebanese propolis.

BACKGROUND: Reactive oxygen species (ROS) are produced by multiple cellular processes and are maintained at optimal levels in normal cells by endogenous antioxidants. In recent years, the search for potential exogenous antioxidants from dietary sources has gained considerable attention to eliminate excess ROS that is associated with oxidative stress related diseases including cancer. Propolis, a resinous honeybee product, has been shown to have protective effects against oxidative stress and anticancer effects against several types of neoplasms. AIM: To investigate the antioxidant and anticancer potential of Lebanese propolis when applied alone or in combination with the promising anticancer compound Thymoquinone (TQ) the main constituent of Nigella sativa essential oil. METHODS: Crude extracts of Lebanese propolis collected from two locations, Rashaya and Akkar-Danniyeh, were prepared in methanol and the total phenolic content was determined by Folin-Ciocalteu method. The antioxidant activity was assessed by the ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and to inhibit H2O2-induced oxidative hemolysis of human erythrocytes. The anticancer activity was evaluated by [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] MTT assay against HCT-116 human colorectal cancer cells and MDA-MB-231 human breast cancer cells. RESULTS: The total phenolic content of propolis extract from Rashaya and Akkar-Danniyeh were 56.81 µg and 83.503 µg of gallic acid equivalent /mg of propolis, respectively. Both natural agents exhibited strong antioxidant activities as evidenced by their ability to scavenge DPPH free radical and to protect erythrocytes against H2O2-induced hemolysis. They also dose-dependently decreased the viability of both cancer cell lines. The IC50 value of each of propolis extract from Rashaya and Akkar-Danniyeh or TQ was 22.3, 61.7, 40.44 µg/mL for breast cancer cells at 72 h and 33.3, 50.9, 33.5 µg/mL for colorectal cancer cells at the same time point, respectively. Importantly, the inhibitory effects of propolis on DPPH radicals and cancer cell viability were achieved at half its concentration when combined with TQ. CONCLUSION: Our results indicate that Lebanese propolis extract has antioxidant and anticancer potential and its combination with TQ could possibly prevent ROS- mediated diseases.

Thymoquinone anticancer activity is enhanced when combined with royal jelly in human breast cancer.

BACKGROUND: Breast cancer is the most common cause of the majority of cancer-related deaths in women, among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has been extensively studied as a potent anticancer molecule against various types of cancers. Honeybee products such as the royal jelly (RJ), the nutritive secretion fed to honeybee queens, exhibit a variety of biological activities besides its anticancer effect. However, the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown. AIM: To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ, RJ, and their combinations in the MDA-MB-231 cell line. METHODS: Cells were treated with TQ, RJ, and their combinations for 24 h. Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we determined the half-maximal inhibitory concentration of TQ. Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions. Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ, RJ, and their combination. Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation. RESULTS: TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration. RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5 µg/mL. High doses of RJ (200 µg/mL) had greater toxicity against MDA-MB-231 cells. Interestingly, the inhibition of cell viability was most pronounced in response to 15 µmol/L TQ and 5 µg/mL RJ. A dose of 15 µmol/L TQ caused a significant increase in the PreG1 population, while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations. TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ. In contrast, no significant regulation of Ki67 expression was observed, indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation. CONCLUSION: This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells, which could confer an advantage for cancer therapy.

Cell Death by Gallotannin Is Associated with Inhibition of the JAK/STAT Pathway in Human Colon Cancer Cells.

BACKGROUND: Gallotannin (GT) is a polyphenol that possesses interesting anticancer properties. However, the mechanisms underlying its antitumor effects have not been well defined. OBJECTIVE: This study was designed to clarify the mechanisms underlying GT antitumor effects in colon cancer cell lines. METHODS: Three isogenic HCT116 cell lines (p53+/+, p53-/-, and p21-/-) were treated with GT for different time points then Western blot, flow cytometry, and senescence analysis were performed to examine the effect of GT on Mitogen-activated protein kinase (MAPK) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) effectors, STAT3 downstream apoptotic targets, Sub-G1 phase, and programmed cell death induction. Transfection using Invitrogen Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, Massachusetts) were used to identify the role of p53 and p21 in the p53-/- and p21-/- cell lines. RESULTS: Both low and high GT concentrations caused MAPKs activation marked by upregulation of extracellular signal-regulated kinase (p-ERK). The preincubation with the antioxidant Tiron (Sigma-Aldrich, St Louis, Missouri) showed that GT's antitumor effects were not mediated by reactive oxygen species. We then examined the effect of GT on the JAK/STAT pathway, which is known to be activated in colorectal cancer. GT totally inhibited the JAK/STAT pathway effectors JAK2, STAT1, and STAT3 and their downstream apoptotic regulators B-cell lymphoma-extra large (Bcl-xL) and c-Myc in all 3 cell lines. HCT116 cancer cells exhibited differential sensitivity to GT with p21-/- cells being the most sensitive and p53+/+ cells that express p21 protein being the least sensitive. In p53+/+ cells, GT induced senescence, whereas in p53-/- and p21-/- cells, GT induced apoptosis in a caspase independent manner marked by Poly(ADP-Ribose) Polymerase (PARP) cleavage, Bcl-2 downregulation, and upregulation of the Bcl-2 associated X (Bax) to B-cell lymphoma 2 (Bcl-2) ratio. In addition, the sub-G1 phase exceeded 50% in p21-/- cells. CONCLUSIONS: Considered together, our results indicate that GT is potent inhibitor of the JAK/STAT pathway in colon cancer irrespective of the p53 and p21 status, which provides insights into its mechanism of anticancer activities and future potential for clinical translation. (Curr Ther Res Clin Exp. 2020; 81:XXX-XXX).

Thymoquinone synergizes with arsenic and interferon alpha to target human T-cell leukemia/lymphoma.

AIMS: To reduce the dose of arsenic used against human T-cell leukemia/lymphoma and to sensitize cells to drug treatment, we combined arsenic/interferon-alpha (As/IFN-α) with thymoquinone (TQ) in HTLV-I positive (HuT-102 and C91) and HTLV-1 negative (CEM and Jurkat) cell lines. MAIN METHODS: Cells were treated with TQ, As/IFN-α and combinations. Trypan blue and flow cytometry were used to investigate viability and cell cycle effects. Annexin-V staining, rhodamine assay and western blotting were used to determine apoptosis induction and changes in protein expression. Efficacy of single drugs and combinations were tested in adult T-cell leukemia (HuT-102) mouse xenograft model. KEY FINDINGS: TQ/As/IFN-α led to a more pronounced and synergistic time-dependent inhibitory effect on HTLV-I positive cells in comparison to As/IFN-α. While As/IFN-α combination was not effective against CEM or Jurkat cells, the triple combination TQ/As/IFN-α sensitized these two cell lines and led to a pronounced time-dependent inhibition of cell viability. TQ/As/IFN-α significantly induced apoptosis in all four cell lines and disrupted the mitochondrial membrane potential. Apoptosis was confirmed by the cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP), downregulation of Bcl-2 and XIAP and upregulation of Bax. TQ alone or in combination activated p53 in HTLV-1 positive cell lines. Strikingly, TQ/As/IFN-α resulted in a pronounced significant decrease in tumor volume in HuT-102 xenograft mouse model, as compared to separate treatments or double combination therapy. SIGNIFICANCE: Our results suggest a strong potential for TQ to enhance the drug targeting effects of the standard clinical drugs As and IFN-α against CD4+ malignant T-cells.

Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells.

Recently, we showed that the metal chelator TPEN targets colon cancer cells through redox cycling of copper. Here, we studied the DNA damage potential of TPEN and deciphered the role of Chk1, ATM and DNA-PK in TPEN-induced toxicity in 3 human colon cancer cell lines, HCT116, SW480 and HT29. We also investigated the role of reactive oxygen species (ROS) in TPEN-induced DNA damage. TPEN reduced cell viability in a dose- and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of γ-H2AX protein and activation of ATM/ATR signaling pathway. Cell death by TPEN was dependent on ROS generation as evidenced by the reversal of cell viability, and DNA damage and the abrogation of γ-H2AX levels in the presence of antioxidants. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10µM). TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression. Cell death by low TPEN concentrations, involved ATM/ATR signaling in all 3 cell lines, since pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells from TPEN-induced DNA damage. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of γ-H2AX and reversed cell death, suggesting that Chk1 and DNA-PK mediate TPEN-induced cytotoxicity in colon cancer cells. This study shows for the first time the involvement of Chk1, DNA-PK and ATM in TPEN-induced DNA damage and confirms our previous findings that ROS generation and the redox cycling of copper in response to TPEN are the main mechanisms by which this compound induces cell death in human colon cancer cells. Inhibition of ATM or DNA-PK did not reverse cytotoxicity at high TPEN concentrations that cause excessive levels of ROS and irreversible cellular damage.

Synthesis, properties, and antitumor effects of a new mixed phosphine gold(I) compound in human colon cancer cells.

The antineoplastic potential of a new stable mixed phosphine gold(I) complex containing tris(tert-butyl)phosphine (tBu3P) and bis(diphenylphosphino)ethene (dppet), namely [Au(tBu3P)(dppet)Cl], has been investigated in the human colon cancer HCT-116 cell line. The (31)P NMR solution study, confirms the structural features observed in the solid state and, in addition, indicates partial formation of dinuclear cationic [Au(tBu3P)2](+) and [Au(dppet)2](+) species. The ionic character and strong Au-P bonds of this gold(I) species are similar to those of the most active antitumor gold compounds so far studied. The title compound was found to exhibit strong cytotoxicity, showing 85 fold greater toxicity than cisplatin (IC50=0.45µM vs IC50=39.16 for cisplatin at 24h) on the HCT-116 line. The cytotoxic effects were, at least partly, mediated by the induction of apoptotic cell death as evidenced by the sub-G1 cell accumulation, oligonucleosomal DNA fragmentation, caspase-3 activation and the release of cytochrome c from the mitochondria. The gold(I) compound showed little interaction with DNA measured through fluorescence quenching studies with calf thymus DNA. The inhibitory effect of the gold(I) compound on intracellular redox proteins has been also observed in pretreated HCT-116 cells. The compound was particularly effective in inhibiting thioredoxin reductase, that is likely responsible for the increased ROS production, and subsequent apoptosis induction via the mitochondrial pathway.