Supplementary figure 1: Hematoxylin and Eosin staining of rat kidney sections with. (a) 50 mg/kg bw Thymoquinone (Tq), (b) 100 mg/kg bw curcumin (Cur) showing no signs of toxicity in the kidney tissues.

Akt, a serine‐threonine protein kinase, is regulated by class‐I PI3K signaling. Akt regulates a wide variety of cell processes including cell proliferation, survival, and angiogenesis through serine/threonine phosphorylation of downstream targets including mTOR and glycogen‐synthase‐kinase‐3‐beta (GSK3β). Targeting cancer‐specific overexpression of Akt protein could be an efficient way to control cancer‐cell proliferation. However, the ATP‐competitive inhibitors are challenged by the highly conserved ATP binding site, and by competition with high cellular concentrations of ATP. We previously developed an allosteric inhibitor, 2‐arylidene‐4, 7‐dimethyl indan‐1‐one (FXY‐1) that showed promising activity against several lung cancer models. In this work, we designed a congeneric series of molecules based on FXY‐1 and optimized lead based on computational, in vitro assays. Computational screening followed by enzyme‐inhibition and cell‐proliferation assays identified a derivative (FCX‐146) as a new lead molecule with threefold greater potency than the parent compound. FCX‐146 increased apoptosis in HL‐60 cells, mediated in part through decreased expression of antiapoptotic Bcl‐2 protein and increased levels of Bax‐2 and Caspase‐3. Molecular‐dynamic simulations showed stable binding of FCX‐146 to an allosteric (i.e., noncatalytic) pocket in Akt. Together, we propose FCX‐146 as a potent second‐generation arylidene indanone compound that binds to the allosteric pocket of Akt and potently inhibits its activation.

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Chronic obstructive pulmonary disease (COPD) is characterized by cigarette smoke‐induced emphysema. Herein, we demonstrate protective effects of Thymoquinone (Tq), an active constituent from Nigella sativa, against cigarette smoke extract (CSE)‐induced abnormalities in bronchial epithelial cells. Dose‐dependent reduction in cell viability was observed in BEAS‐2B cells when exposed to different CSE concentrations, which was significantly reversed by Tq evident by LDH release. Levels of SOD, CAT, GR, GSH, and mitochondrial membrane ATPases were significantly reduced upon CSE exposure, an event, again, antagonized in presence of Tq. Similarly, Tq treatment significantly blocked CSE‐induced 4HNE elevations. Further, Tq‐improved mitochondrial dysfunction caused by CSE and significantly decreased autophagy/mitophagy markers like LC3II and p‐Drp. Tq also reduced necroptosis markers such as p‐MLKL, RIP‐1, and RIP‐3, by stabilizing PINK‐1 levels. In summary, Tq possesses protective properties against human bronchial epithelial cell autophagy/mitophagy‐dependent necroptosis caused by CSE, which warrants considerable attention for further preclinical evaluations. Practical applications This study demonstrates Thymoquinone (Tq), a natural plant extract to possess protective properties against human bronchial epithelial cell autophagy/mitophagy‐dependent necroptosis caused by cigarette smoke extract. The demonstrated efficacy of Tq will throw light for further preclinical evaluation of this molecule in CSE‐mediated complications. A detailed in vivo research is recommended.

Interleukin (IL)‐7 acts via the IL‐7 receptor in metastatic tumor progression in prostate cancer (PC). The current study aimed to evaluate thymoquinone (Tq), an active constituent from Nigella sativa against IL‐7–driven tumor progression and metastatic invasion in PC cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to assess the proliferation of PC cells. Enzyme‐linked immunosorbent assay was used to detect the expression of IL‐7 and matrix metalloproteinases (MMPs). Tumor‐cell transendothelial, scratch wound and cell scatter assays were performed to mimic metastasis. Western immunoblotting was used to measure the level of proteins. Tq effectively controlled the proliferation of DU‐145, PC‐3, and LNCaP cells with GI50 of 10.18, 12.40, and 16.78 µM, respectively. IL‐7 and IL‐7R were natively expressed in all PC types, while maximal expression was detected in DU‐145. IL‐7 promoted metastatic events, such as transendothelial migration, cell scatter, and cell invasion of DU‐145 cells in a dose‐dependent manner that was inhibited by Tq. Furthermore, Tq also downregulated p‐Akt and NF‐κB in DU‐145 cells induced by IL‐7 antibody and reduced the levels of MMP‐3 and MMP‐7 in these cells in a dose‐dependent manner. Collectively, Tq has excellent efficacy in controlling tumor progression, migration, and invasion of DU‐145 cells that were driven by the activation of MMPs through IL‐7/Akt/NF‐κB signaling.