6-n-Butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene Improves the X-ray Sensitivity on Inhibiting Proliferation and Promoting Oxidative Stress and Apoptosis of Oral Cancer Cells.

This in vitro study examines the anti-oral cancer effects and mechanisms of a combined X-ray/SK2 treatment, i.e., X-ray and 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene (SK2). ATP cell viability and flow cytometry-based cell cycle, apoptosis, oxidative stress, and DNA damage assessments were conducted. The X-ray/SK2 treatment exhibited lower viability in oral cancer (Ca9-22 and CAL 27) cells than in normal (Smulow-Glickman, S-G) cells, i.e., 32.0%, 46.1% vs. 59.0%, which showed more antiproliferative changes than with X-ray or SK2 treatment. Oral cancer cells under X-ray/SK2 treatment showed slight subG1 and G2/M increments and induced high annexin V-monitored apoptosis compared to X-ray or SK2 treatment. The X-ray/SK2 treatment showed higher caspase 3 and 8 levels for oral cancer cells than other treatments. X-ray/SK2 showed a higher caspase 9 level in CAL 27 cells than other treatments, while Ca9-22 cells showed similar levels under X-ray and/or SK2. The X-ray/SK2 treatment showed higher reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) depletion than other treatments. Meanwhile, the mitochondrial superoxide (MitoSOX) and glutathione levels in X-ray/SK2 treatment did not exhibit the highest rank compared to others. Moreover, oral cancer cells had higher γH2AX and/or 8-hydroxy-2-deoxyguanosine levels from X-ray/SK2 treatment than others. All these measurements for X-ray/SK2 in oral cancer cells were higher than in normal cells and attenuated by N-acetylcysteine. In conclusion, X-ray/SK2 treatment showed ROS-dependent enhanced antiproliferative, apoptotic, and DNA damage effects in oral cancer cells with a lower cytotoxic influence on normal cells.

Synergistic Antiproliferation of Cisplatin and Nitrated [6,6,6]Tricycle Derivative (SK2) for a Combined Treatment of Oral Cancer Cells.

SK2, a nitrated [6,6,6]tricycle derivative with an n-butyloxy group, showed selective antiproliferation effects on oral cancer but not on normal oral cells. This investigation assessed for the first time the synergistic antiproliferation potential of cisplatin/SK2 in oral cancer cells. Cell viability assay at 24 h showed that a low dose of combined cisplatin/SK2 (10 µM/10 µg/mL) provided more antiproliferation than cisplatin or SK2 alone. Cisplatin/SK2 triggered also more apoptosis inductions in terms of subG1 accumulation, annexin V, pancaspase, and caspase 3/8/9 measurements. Moreover, cisplatin/SK2 provided more oxidative stress and DNA damage in oral cancer cells than independent treatments. Oxidative stress inhibitors rescued the cisplatin/SK2-induced antiproliferation and oxidative stress generation. Moreover, cisplatin/SK2 induced more antiproliferation, apoptosis, oxidative stress, and DNA damage in oral cancer cells than in normal oral cells (S-G). In conclusion, low-dose cisplatin/SK2 combined treatment promoted selective and synergistic antiproliferation in oral cancer cells depending on oxidative-stress-associated responses.

Combined Treatment of Nitrated [6,6,6]Tricycles Derivative (SK2)/Ultraviolet C Highly Inhibits Proliferation in Oral Cancer Cells In Vitro.

Combined treatment is an effective strategy to improve anticancer therapy, but severe side effects frequently limit this application. Drugs inhibiting the proliferation of cancer cells, but not normal cells, display preferential antiproliferation to cancer cells. It shows the benefits of avoiding side effects and enhancing antiproliferation for combined treatment. Nitrated [6,6,6]tricycles derivative (SK2), a novel chemical exhibiting benzo-fused dioxabicyclo[3.3.1]nonane core with an n-butyloxy substituent, exhibiting preferential antiproliferation, was chosen to evaluate its potential antioral cancer effect in vitro by combining it with ultraviolet C (UVC) irradiation. Combination treatment (UVC/SK2) caused lower viability in oral cancer cells (Ca9-22 and OC-2) than single treatment (20 J/m2 UVC or 10 µg/mL SK2), i.e., 42.3%/41.1% vs. 81.6%/69.2%, and 89.5%/79.6%, respectively. In contrast, it showed a minor effect on cell viability of normal oral cells (HGF-1), ranging from 82.2 to 90.6%. Moreover, UVC/SK2 caused higher oxidative stress in oral cancer cells than normal cells through the examination of reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential. UVC/SK2 also caused subG1 increment associated with apoptosis detections by assessing annexin V; panaspase; and caspases 3, 8, and 9. The antiproliferation and oxidative stress were reverted by N-acetylcysteine, validating the involvement of oxidative stress in antioral cancer cells. UVC/SK2 also caused DNA damage by detecting γH2AX and 8-hydroxy-2'-deoxyguanosine in oral cancer cells. In conclusion, SK2 is an effective enhancer for improving the UVC-caused antiproliferation against oral cancer cells in vitro. UVC/SK2 demonstrated a preferential and synergistic antiproliferation ability towards oral cancer cells with little adverse effects on normal cells.

Antiproliferation- and Apoptosis-Inducible Effects of a Novel Nitrated [6,6,6]Tricycle Derivative (SK2) on Oral Cancer Cells.

The benzo-fused dioxabicyclo[3.3.1]nonane core is the central framework in several natural products. Using this core, we had developed a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group, namely, SK2. The anticancer potential of SK2 was not assessed. This study aimed to determine the antiproliferative function and investigated possible mechanisms of SK2 acting on oral cancer cells. SK2 preferentially killed oral cancer cells but caused no harmful effect on non-malignant oral cells. After the SK2 exposure of oral cancer cells, cells in the sub-G1 phase accumulated. This apoptosis-like outcome of SK2 treatment was validated to be apoptosis via observing an increasing annexin V population. Mechanistically, apoptosis signalers such as pancaspase, caspases 8, caspase 9, and caspase 3 were activated by SK2 in oral cancer cells. SK2 induced oxidative-stress-associated changes. Furthermore, SK2 caused DNA damage (γH2AX and 8-hydroxy-2'-deoxyguanosine). In conclusion, a novel nitrated [6,6,6]tricycle-derived compound, SK2, exhibits a preferential antiproliferative effect on oral cancer cells, accompanied by apoptosis, oxidative stress, and DNA damage.

Combined Treatment with Cryptocaryone and Ultraviolet C Promotes Antiproliferation and Apoptosis of Oral Cancer Cells.

Cryptocaryone (CPC) was previously reported as preferential for killing natural products in oral cancer cells. However, its radiosensitizing potential combined with ultraviolet C (UVC) cell killing of oral cancer cells remains unclear. This study evaluates the combined anti-proliferation effect and clarifies the mechanism of combined UVC/CPC effects on oral cancer cells. UVC/CPC shows higher anti-proliferation than individual and control treatments in a low cytotoxic environment on normal oral cells. Mechanistically, combined UVC/CPC generates high levels of reactive oxygen species and induces mitochondrial dysfunction by generating mitochondrial superoxide, increasing mitochondrial mass and causing the potential destruction of the mitochondrial membrane compared to individual treatments. Moreover, combined UVC/CPC causes higher G2/M arrest and triggers apoptosis, with greater evidence of cell cycle disturbance, annexin V, pancaspase, caspases 3/7 expression or activity in oral cancer cells than individual treatments. Western blotting further indicates that UVC/CPC induces overexpression for cleaved types of poly (ADP-ribose) polymerase and caspase 3 more than individual treatments. Additionally, UVC/CPC highly induces γH2AX and 8-hydroxy-2'-deoxyguanosine adducts as DNA damage in oral cancer cells. Taken together, CPC shows a radiosensitizing anti-proliferation effect on UVC irradiated oral cancer cells with combined effects through oxidative stress, apoptosis and DNA damage.

Regulatory effects of noncoding RNAs on the interplay of oxidative stress and autophagy in cancer malignancy and therapy.

Noncoding RNAs (ncRNAs) regulation of various diseases including cancer has been extensively studied. Reactive oxidative species (ROS) elevated by oxidative stress are associated with cancer progression and drug resistance, while autophagy serves as an ROS scavenger in cancer cells. However, the regulatory effects of ncRNAs on autophagy and ROS in various cancer cells remains complex. Here, we explore how currently investigated ncRNAs, mainly miRNAs and lncRNAs, are involved in ROS production through modulating antioxidant genes. The regulatory effects of miRNAs and lncRNAs on autophagy-related (ATG) proteins to control autophagy activity in cancer cells are discussed. Moreover, differential expression of ncRNAs in tumor and normal tissues of cancer patients are further analyzed using The Cancer Genome Atlas (TCGA) database. This review hypothesizes links between ATG genes- or antioxidant genes-modulated ncRNAs and ROS production, which might result in tumorigenesis, malignancy, and cancer recurrence. A better understanding of the regulation of ROS and autophagy by ncRNAs might advance the use of ncRNAs as diagnostic and prognostic markers as well as therapeutic targets in cancer therapy.

Soft Coral-Derived Dihydrosinularin Exhibits Antiproliferative Effects Associated with Apoptosis and DNA Damage in Oral Cancer Cells.

Dihydrosinularin (DHS) is an analog of soft coral-derived sinularin; however, the anticancer effects and mechanisms of DHS have seldom been reported. This investigation examined the antiproliferation ability and mechanisms of DHS on oral cancer cells. In a cell viability assay, DHS showed growth inhibition against several types of oral cancer cell lines (Ca9-22, SCC-9, OECM-1, CAL 27, OC-2, and HSC-3) with no cytotoxic side effects on non-malignant oral cells (HGF-1). Ca9-22 and SCC-9 cell lines showing high susceptibility to DHS were selected to explore the antiproliferation mechanisms of DHS. DHS also causes apoptosis as detected by annexin V, pancaspase, and caspase 3 activation. DHS induces oxidative stress, leading to the generation of reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) and mitochondrial membrane potential (MitoMP) depletion. DHS also induced DNA damage by probing γH2AX phosphorylation. Pretreatment with the ROS scavenger N-acetylcysteine (NAC) can partly counter these DHS-induced changes. We report that the marine natural product DHS can inhibit the cell growth of oral cancer cells. Exploring the mechanisms of this cancer cell growth inhibition, we demonstrate the prominent role DHS plays in oxidative stress.

Nepenthes Ethyl Acetate Extract Provides Oxidative Stress-Dependent Anti-Leukemia Effects.

Several kinds of solvents have been applied to Nepenthes extractions exhibiting antioxidant and anticancer effects. However, they were rarely investigated for Nepenthes ethyl acetate extract (EANT), especially leukemia cells. The purpose of the present study was to evaluate the antioxidant properties and explore the antiproliferation impact and mechanism of EANT in leukemia cells. Five standard assays demonstrated that EANT exhibits antioxidant capability. In the cell line model, EANT dose-responsively inhibited cell viabilities of three leukemia cell lines (HL-60, K-562, and MOLT-4) based on 24 h MTS assays, which were reverted by pretreating oxidative stress and apoptosis inhibitors (N-acetylcysteine and Z-VAD-FMK). Due to similar sensitivities among the three cell lines, leukemia HL-60 cells were chosen for exploring antiproliferation mechanisms. EANT caused subG1 and G1 cumulations, triggered annexin V-detected apoptosis, activated apoptotic caspase 3/7 activity, and induced poly ADP-ribose polymerase expression. Moreover, reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization were generated by EANT, which was reverted by N-acetylcysteine. The antioxidant response to oxidative stress showed that EANT upregulated mRNA expressions for nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), thioredoxin (TXN), heme oxygenase 1 (HMOX1), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. Moreover, these oxidative stresses led to DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) and were alleviated by N-acetylcysteine. Taken together, EANT demonstrated oxidative stress-dependent anti-leukemia ability to HL-60 cells associated with apoptosis and DNA damage.

Comparison of Antioxidant and Anticancer Properties of Soft Coral-Derived Sinularin and Dihydrosinularin.

Marine natural products are abundant resources for antioxidants, but the antioxidant property of the soft corals-derived sinularin and dihydrosinularin were unknown. This study aimed to assess antioxidant potential and antiproliferation effects of above compounds on cancer cells, and to investigate the possible relationships between them. Results show that sinularin and dihydrosinularin promptly reacted with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), and hydroxyl (•OH), demonstrating a general radical scavenger activity. Sinularin and dihydrosinularin also show an induction for Fe+3-reduction and Fe+2-chelating capacity which both strengthen their antioxidant activities. Importantly, sinularin shows higher antioxidant properties than dihydrosinularin. Moreover, 24 h ATP assays show that sinularin leads to higher antiproliferation of breast, lung, and liver cancer cells than dihydrosinularin. Therefore, the differential antioxidant properties of sinularin and dihydrosinularin may contribute to their differential anti-proliferation of different cancer cells.

Combined Treatment of Sulfonyl Chromen-4-Ones (CHW09) and Ultraviolet-C (UVC) Enhances Proliferation Inhibition, Apoptosis, Oxidative Stress, and DNA Damage against Oral Cancer Cells.

The sensitizing effect of chromone-derived compounds on UVC-induced proliferation inhibition has not been comprehensively investigated so far. The subject of this study was to examine the proliferation change of oral cancer cells while using the combined treatment of UVC (254 nm) with our previously developed sulfonyl chromen-4-ones (CHW09), namely UVC/CHW09. Cell viability, apoptosis, oxidative stress, and DNA damage for the individual and combined treatments for UVC and/or CHW09 were examined in oral cancer Ca9-22 cells. In 24 h MTS assay, UVC (30 J/m2; UVC30), or CHW09 (25 and 50 µg/mL; namely, CHW09-25 and CHW09-50) show 54%, 59%, and 45% viability. The combined treatment (UVC30/CHW09-25 and UVC30/CHW09-50) show lower cell viability (45% and 35%). Mechanistically, UVC/CHW09 induced higher apoptosis than individual treatments and untreated control, which were supported by the evidence of flow cytometry for subG1, annexin V/7-aminoactinomycin D, pancaspase and caspases 3/7 activity, and western blotting for cleaved poly(ADP-ribose) polymerase. Moreover, this cleaved PARP expression was downregulated by pancaspase inhibitor Z-VAD-FMK. UVC/CHW09 showed higher oxidative stress than individual treatments and untreated control in terms of flow cytometry for reactive oxygen species, mitochondrial membrane potential, and mitochondrial mass. Furthermore, UVC/CHW09 showed higher DNA damage than individual treatments and untreated control in terms of flow cytometry for H2A histone family member X and 8-oxo-2'-deoxyguanosine. In conclusion, combined treatment UVC/CHW09 suppresses proliferation, and promotes apoptosis, oxidative stress, and DNA damage against oral cancer cells, providing a novel application of sulfonyl chromen-4-ones in order to sensitize UVC induced proliferation inhibition for oral cancer therapy.

Ethyl acetate extract of Nepenthes adrianii x clipeata induces antiproliferation, apoptosis, and DNA damage against oral cancer cells through oxidative stress.

Nepenthes plants are regarded as a kind of Traditional Chinese Medicine for several diseases but its anticancer activity remain unclear. The subject of this study is to evaluate the antiproliferation effects on oral cancer cells by Nepenthes plants using ethyl acetate extract of Nepenthes adrianii x clipeata (EANA). Cell viability was detected using MTS assay. Its detailed mechanisms including cell cycle, apoptosis, oxidative stress, and DNA damage were explored by flow cytometry or western blotting. For 24 hours EANA treatment, five kinds of oral cancer cells (CAL 27, Ca9-22, OECM-1, HSC-3, and SCC9) show IC50 values of cell viability ranging from 8 to 17 µg/mL but the viability of normal oral cells (HGF-1) remains over 80%. Subsequently, CAL 27 and Ca9-22 cells with high sensitivity to EANA were chosen to investigate the detailed mechanism. EANA displays the time course and concentration effects for inducing apoptosis based on flow cytometry (subG1 and annexin V analyses) and western blotting [cleaved poly (ADP-ribose) polymerase (c-PARP)]. Oxidative stress and DNA damage were induced by EANA treatments in oral cancer cells through reactive oxygen species (ROS), mitochondrial membrane potential disruption, mitochondrial superoxide, and γH2AX. All these changes of EANA treatments in oral cancer cells were reverted by the ROS scavenger N-acetylcysteine pretreatment. Therefore, EANA induces preferential killing, apoptosis, and DNA damage against oral cancer cells through oxidative stress.

Sulfonyl chromen-4-ones (CHW09) shows an additive effect to inhibit cell growth of X-ray irradiated oral cancer cells, involving apoptosis and ROS generation.

Purpose: This study evaluates the growth inhibiting potential of our previously described sulfonyl chromen-4-ones (CHW09) compound in X-ray irradiated oral cancer cells. Materials and methods: The growth inhibiting effect and mechanism of combined CHW09/X-ray treatment was examined by analyzing cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), and DNA damage. Results: Individual treatments of CHW09 (10 µg/mL) and X-ray irradiation (12 Gy) slightly decreased cell viability of oral cancer Ca9-22 (87.25% and 86.54%) and CAL 27 (80.00% and 74.01%) cells and normal oral HGF-1 cells (92.76% and 87.56%) at 24 h-MTS assay, respectively. In a combined treatment (CHW09/X-ray), the cell viability in Ca9-22 and CAL 27 cells was significantly decreased to 73.48% and 59.07%, whereas HGF-1 cells maintained 84.97% viability in 24 h-MTS assay. For CAL 27 cells, both 72 h-MTS assay and clonogenic assay showed that CHW09/X-ray resulted in more growth inhibition than other treatments. Intracellular ROS levels of CHW09/X-ray were higher than for CHW09, X-ray and control. CHW09/X-ray and X-ray alone had higher G2/M arrest than the control and CHW09 alone. Moreover, flow cytometry and western blotting showed that CHW09/X-ray treatment caused higher apoptosis levels. Levels of H2A histone family member X (γH2AX)-based DNA damage and 8-oxo-2'-deoxyguanosine (8-oxodG)-oxidative DNA damage of CHW09/X-ray were higher than for CHW09, X-ray and control. Conclusion: CHW09/X-ray treatment had additive growth inhibiting effects against X-ray irradiated oral cancer cells, partly attributing to apoptosis and ROS generation.

A novel sulfonyl chromen-4-ones (CHW09) preferentially kills oral cancer cells showing apoptosis, oxidative stress, and DNA damage.

Several functionalized chromones, the key components of naturally occurring oxygenated heterocycles, have anticancer effects but their sulfone compounds are rarely investigated. In this study, we installed a sulfonyl substituent to chromen-4-one skeleton and synthesized CHW09 to evaluate its antioral cancer effect in terms of cell viability, cell cycle, apoptosis, oxidative stress, and DNA damage. In cell viability assay, CHW09 preferentially kills two oral cancer cells (Ca9-22 and CAL 27), less affecting normal oral cells (HGF-1). Although CHW09 does not change the cell cycle distribution significantly, CHW09 induces apoptosis validated by flow cytometry for annexin V and by western blotting for cleaved poly(ADP-ribose) polymerase (PARP), and caspases 3/8/9. These apoptosis signaling expressions are partly decreased by apoptosis inhibitor (Z-VAD-FMK) or free radical scavenger (N-acetylcysteine). Furthermore, CHW09 induces oxidative stress validated by flow cytometry for the generations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), and the suppression of mitochondrial membrane potential (MMP). CHW09 also induces DNA damage validated by flow cytometry for the increases of DNA double strand break marker γH2AX and oxidative DNA damage marker 8-oxo-2'-deoxyguanosine (8-oxodG). Therefore, our newly synthesized CHW09 induces apoptosis, oxidative stress, and DNA damage, which may lead to preferential killing of oral cancer cells compared with normal oral cells.

Antioxidant Properties of Fractions for Unripe Fruits of Capsicum annuum L. var. Conoides.

BACKGROUND: Capsicum plant, especially for C. annuum, is an abundant resource for bioactive antioxidants, but few studies have examined the unripe fruit part of the Capsicum plant. OBJECTIVE: MeOH extract of unripe fruits of C. annuum L. var. conoides (UFCA) was chromatographed over a silica gel column using a gradient of CH2Cl2/MeOH as eluent to produce 9 fractions. Antioxidant activities are evaluated along with cell viabilities of 9 fractions of UFCA. METHOD: The antioxidant properties were analyzed in terms of total phenol content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2-azinobis (3-ethyl-benzothiazoline-6- sulfonic acid) (ABTS) radical scavenging, ferric reducing, and ferrous ion-chelating ability. The cell viability of human oral cancer cells (Ca9-22) was measured by 3-(4,5-dimethylthiazol-2-yl)-(3-carboxymethoxyphenyl)-2- (4-sulphophenyl)-2H-tetrazolium (MTS) assay. RESULTS: Except for TFC, fractions (Frs.) 1 and 2 showed the lowest level of these antioxidant properties. Frs. 3 to 9 showed dose-responsive induction for antioxidant effects. Fr. 8 and Fr. 5 respectively showed the highest levels of TPC and TFC for 1162 ± 11 gallic acid equivalents (GAE) (mg)/UFCA (g) and 1295 ± 32 quercetin equivalents (QCE) (mg)/UFCA (g). The cell viability of Fr. 3 was moderately decreased (78.2%) while those of Frs. 4, 5, and 9 were dramatically decreased (55.6, 57.8, and 46.8%, respectively) in oral cancer Ca9-22 cells. UFCA-derived 14 compounds/mixtures derived from Frs. 1, 2, 3, 4, and 8 displayed differential antioxidant performance for these analyses. CONCLUSION: Taken together, fractions of UFCA displayed diverse antioxidant and anticancer effects for oral cancer cells. Some fractions of UFCA may be potent natural antioxidant supplements for antioral cancer cell treatment.