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Oral squamous cell carcinoma (OSCC) is a worldwide public health problem, accounting for approximately 90% of all oral cancers, and is the eighth most common cancer in men. Cisplatin and carboplatin are the main chemotherapy drugs used in the clinic. However, in addition to their serious side effects, such as damage to the nervous system and kidneys, there is also drug resistance. Thus, the development of new drugs becomes of great importance. Naphthoquinones have been described with antitumor activity. Some of them are found in nature, but semi synthesis has been used as strategy to find new chemical entities for the treatment of cancer. In the present study, we promote a multiple component reaction (MCR) among lawsone, arylaldehydes, and benzylamine to produce sixteen chemoselectively derivated Mannich adducts of 1,4-naphthoquinones in good yield (up to 97%). The antitumor activities and molecular mechanisms of action of these compounds were investigated in OSCC models and the compound 6a induced cytotoxicity in three different tumor cell lines (OSCC4, OSCC9, and OSCC25) and was more selective (IS > 2) for tumor cells than the chemotropic drug carboplatin and the controls lapachol and shikonin, which are chemically similar compounds with cytotoxic effects. The 6a selectively and significantly reduced the amount of cell colony growth, was not hemolytic, and tolerable in mice with no serious side effects at a concentration of 100 mg/kg with a LD50 of 150 mg/kg. The new compound is biologically stable with a profile similar to carboplatin. Morphologically, 6a does not induce cell retraction or membrane blebs, but it does induce intense vesicle formation and late emergence of membrane bubbles. Exploring the mechanism of cell death induction, compound 6a does not induce ROS formation, and cell viability was not affected by inhibitors of apoptosis (ZVAD) and necroptosis (necrostatin 1). Autophagy followed by a late apoptosis process appears to be the death-inducing pathway of 6a, as observed by increased viability by the autophagy inhibitor (3-MA) and by the appearance of autophagosomes, later triggering a process of late apoptosis with the presence of caspase 3/7 and DNA fragmentation. Molecular modeling suggests the ability of the compound to bind to topoisomerase I and II and with greater affinity to hPKM2 enzyme than controls, which could explain the mechanism of cell death by autophagy. Finally, the in-silico prediction of drug-relevant properties showed that compound 6a has a good pharmacokinetic profile when compared to carboplatin and doxorubicin. Among the sixteen naphthoquinones tested, compound 6a was the most effective and is highly selective and well tolerated in animals. The induction of cell death in OSCC through autophagy followed by late apoptosis possibly via inhibition of the PKM2 enzyme points to a promising potential of 6a as a new preclinical anticancer candidate.

Oral squamous cell carcinoma (OSCC) is a global public health problem with high incidence and mortality. The chemotherapeutic agents used in the clinic, alone or in combination, usually lead to important side effects. Thus, the discovery and development of new antineoplastic drugs are essential to improve disease prognosis and reduce toxicity. In the present study, acridine-core naphthoquinone compounds were synthesized and evaluated for their antitumor activity in OSCC cells. The mechanism of action, pharmacokinetics, and toxicity parameters of the most promising compound was further analyzed using in silico, in vitro, and in vivo methods. Among the derivatives, compound 4e was highly cytotoxic (29.99 µM) and selective (SI 2.9) at levels comparable and generally superior to chemotherapeutic controls. Besides, compound 4e proved to be non-hemolytic, stable, and well tolerated in animals at all doses tested. Mechanistically, compound 4e promoted cell death by apoptosis in the OSCC cell, and molecular docking studies suggested this compound possibly targets enzymes important for tumor progression, such as RSK2, PKM2, and topoisomerase IIα. Importantly, compound 4e presented a pharmacological profile within desirable parameters for drug development, showing promise for future preclinical trials.

The oral squamous cell carcinoma (OSCC) stands out as a public health problem due to its high incidence and low survival rate, despite advances in diagnosis and treatment. Moreover, the most commonly chemotherapeutic agents for OSCC, such as carboplatin and cisplatin, generate important side effects, evidencing the urgency in developing new drugs. Naphthoquinones are an important class of natural products or synthetic compounds with cytotoxic effect demonstrated on different cancer types. In the present study, thirty-five 1,4-naphthoquinones tethered to 1,2,3-1H-triazoles were synthesized and the antitumor activity and molecular mechanisms were evaluated in several assays including in vitro and in vivo models of OSCC and normal oral human cells. Compounds 16a, 16b and 16 g were able to induce cytotoxicity in three different tumor cell lines of human OSCC (SCC4, SCC9 and SCC25) and were more toxic and selective to tumor cells (Selective Index, SI > 2) than classical and chemically similar controls (Carboplatin and Lapachol). Compound 16 g showed the higher SI value. Besides, compounds 16a, 16b and 16 g significantly reduced colony formation of SCC9 cells in the tested concentrations. Hemolytic assay using compounds 16a, 16b and 16 g at high concentrations showed no compound exhibited hemolysis higher than 5%, similar to controls. In vivo acute toxicity study showed that 16 g was the only one, among the three compounds, with no apparent limiting toxic effects on mice in the tested concentrations. Thus, the investigation of cell death mechanisms was conducted with this compound. 16 g does not trigger ROS production nor binds to DNA. On the other hand, compound 16 g induced microtubule disorganization, and molecular modeling studies suggests a potential mechanism of action related to inhibition of topoisomerases and/or hPKM2 activities. Cell morphology, pyknotic nuclei presence, cleaved caspase-3 staining and viability assays using caspase-3 inhibitors demonstrate compound 16 g induced cell death through apoptosis. Among the 35 synthesized triazole naphthoquinones, compound 16 g was the most effective compound against OSCC cells, presenting high cytotoxicity (~35 µM), selectivity (SI ~ 6) and low acute toxicity on animals, and therefore might be considered for future cancer therapy., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells., Methods: The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry., Results: We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling., Conclusion: From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.