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Increased neddylation benefits the survival of several types of cancer cells. The inhibition of neddylation has the potential to exert anticancer effects but is rarely assessed in oral cancer cells. This study aimed to investigate the antiproliferation potential of a neddylation inhibitor MLN4924 (pevonedistat) for oral cancer cells. MLN4924 inhibited the cell viability of oral cancer cells more than that of normal oral cells (HGF-1) with 100% viability, that is, IC 50 values of oral cancer cells (CAL 27, OC-2, and Ca9-22) are 1.8, 1.4, and 1.9 μM. MLN4924 caused apoptotic changes such as the subG1 accumulation, activation of annexin V, pancaspase, and caspases 3/8/9 of oral cancer cells at a greater rate than in normal oral cells. MLN4924 induced greater oxidative stress in oral cancer cells compared to normal cells by upregulating reactive oxygen species and mitochondrial superoxide and depleting the mitochondrial membrane potential and glutathione. In oral cancer cells, preferential inductions also occurred for DNA damage (γH2AX and 8-oxo-2'-deoxyguanosine). Therefore, this investigation demonstrates that MLN4924 is a potential anti-oral-cancer agent showing preferential inhibition of apoptosis and promotion of DNA damage with fewer cytotoxic effects on normal cells., (© 2023 Wiley Periodicals LLC.)

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.

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.

We previously found that marine sponge-derived manoalide induced antiproliferation and apoptosis of oral cancer cells as well as reactive species generations probed by dichloro-dihydrofluorescein diacetate (DCFH-DA) and MitoSOX Red. However, the sources of cellular and mitochondrial redox stresses and the mutual interacting effects between these redox stresses and apoptosis remain unclear. To address this issue, we examined a panel of reactive species and used the inhibitors of cellular reactive species ( N -acetylcysteine (NAC)), mitochondrial reactive species (MitoTEMPO), and apoptosis (Z-VAD-FMK; ZVAD) to explore their interactions in manoalide-treated oral cancer Ca9-22 and CAL 27 cells. Hydroxyl (˙OH), nitrogen dioxide (NO 2 ˙), nitric oxide (˙NO), carbonate radical-anion (CO 3 ˙- ), peroxynitrite (ONOO - ), and superoxide (O 2 ˙- ) were increased in oral cancer cells following manoalide treatments in terms of fluorescence staining and flow cytometry. Cellular reactive species (˙OH, NO 2 · ) were induced in oral cancer cells following manoalide treatment for 6 h. NAC, MitoTEMPO, and ZVAD inhibit manoalide-induced apoptosis in terms of annexin V and pancaspase activity assays. Moreover, NAC inhibits mitochondrial reactive species and MitoTEMPO inhibits cellular reactive species, suggesting that cellular and mitochondrial reactive species can crosstalk to regulate each other. ZVAD shows suppressing effects on the generation of both cellular and mitochondrial reactive species. In conclusion, manoalide induces reciprocally activation between cellular and mitochondrial reactive species and apoptosis in oral cancer cells.3 ˙- , and ONOO - ) as well as cellular and mitochondrial reactive species (O 2 ˙- ) were induced in oral cancer cells following manoalide treatment for 6 h. NAC, MitoTEMPO, and ZVAD inhibit manoalide-induced apoptosis in terms of annexin V and pancaspase activity assays. Moreover, NAC inhibits mitochondrial reactive species and MitoTEMPO inhibits cellular reactive species, suggesting that cellular and mitochondrial reactive species can crosstalk to regulate each other. ZVAD shows suppressing effects on the generation of both cellular and mitochondrial reactive species. In conclusion, manoalide induces reciprocally activation between cellular and mitochondrial reactive species and apoptosis in oral cancer cells., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Hui-Ru Wang et al.)

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/m 2 ; 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.

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.

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., (© 2018 Wiley Periodicals, Inc.)

We have previously found that the aqueous extract of Gracilaria tenuistipitata (AEGT) and its partitioned fractions had antioxidant properties in biochemical assays. Although the butanol-partitioned fraction of AEGT (AEGT-pBuOH) had a stronger antioxidant performance than AEGT, its biological effects are still unknown. In this study, the cellular responses of oral cancer cells to AEGT-pBuOH were monitored in terms of cell viability, cell cycle progression, apoptosis, and oxidative stress responses. In an ATP content assay, the cell viability of oral cancer cells treated with AEGT-pBuOH was dose responsively inhibited (p < 0.005). For flow cytometry, AEGT-pBuOH was also found to dose responsively induce cell cycle disturbance by propidium iodide (PI) staining and to induce apoptosis by annexin V/PI and pan-caspase staining (p < 0.005). In AEGT-pBuOH-treated oral cancer cells, the reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased in a dose-response manner (p < 0.005). These results suggest that AEGT-pBuOH inhibited the proliferation and induced apoptosis of oral cancer cells involving the ROS generation and mitochondrial depolarization.

Roe protein hydrolysates were reported to have antioxidant property but the anticancer effects were less addressed, especially for oral cancer. In this study, we firstly used the ultrafiltrated roe hydrolysates (URH) derived from giant grouper (Epinephelus lanceolatus) to evaluate the impact of URH on proliferation against oral cancer cells. We found that URH dose-responsively reduced cell viability of two oral cancer cells (Ca9-22 and CAL 27) in terms of ATP assay. Using flow cytometry, URH-induced apoptosis of Ca9-22 cells was validated by morphological features of apoptosis, sub-G1 accumulation, and annexin V staining in dose-responsive manners. URH also induced oxidative stress in Ca9-22 cells in terms of reactive oxygen species (ROS)/superoxide generations and mitochondrial depolarization. Taken together, these data suggest that URH is a potential natural product for antioral cancer therapy.

Background: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer., Methods: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage., Results: High concentrations (50-400 μg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 μg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations., Conclusions: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Many red algae-derived natural products are known to have anticancer effects. The biological functions of the red alga Solieria robusta from the Karachi coast (Pakistan) remain unclear. Here, we prepared a methanolic extracts of S. robusta (MESR) to examine its possible anti-oral cancer effects and the corresponding mechanism of action. Cell viability of MESR-incubated oral cancer Ca9-22 cells was dose-responsively decreased (p<0.001). According to a propidium iodide (PI)-based assay the cell cycle distribution was dramatically changed, especially for subG1 accumulation. Annexin V/PI assay of apoptosis using flow cytometry also showed that MESR-incubated Ca9-22 cells were dose-responsively increased (p<0.001). For evaluation of oxidative stress in MESR-incubated Ca9-22 cells, we found that reactive oxygen species (ROS) were overexpressed dose- and time-responsively and mitochondrial depolarization was also increased (p<0.001). Taken together, MESR showed inhibitory effects on oral cancer proliferation coupled with apoptosis and oxidative stress.

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.

Background: Methanolic extracts of Gracilaria tenuistipitata (MEGT) were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses., Methods: In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated., Results: The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose-response manner (p < 0.05). The sub-G1 population and annexin V intensity of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.0005 and p < 0.001, respectively). The γH2AX intensities of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.05). The reactive oxygen species (ROS) and glutathione (GSH)-positive intensities of MEGT-treated Ca9-22 oral cancer cells were significantly increased and decreased, respectively, in a dose-response manner (p < 0.05). The DiOC2(3) intensity for mitochondrial membrane potential (MMP) of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose-response manner (p < 0.05)., Conclusions: These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC).

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.

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. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is insensitive to target therapy for non-TNBC and needs novel drug discovery. Extracts of the traditional herb Boesenbergia plant in Southern Asia exhibit anticancer effects and contain novel bioactive compounds but merely show cytotoxicity. We recently isolated a new compound from B. stenophylla, stenophyllol B (StenB), but the impact and mechanism of its proliferation-modulating function on TNBC cells remain uninvestigated. This study aimed to assess the antiproliferative responses of StenB in TNBC cells and examine the drug safety in normal cells. StenB effectively suppressed the proliferation of TNBC cells rather than normal cells in terms of an ATP assay. This preferential antiproliferative function was alleviated by pretreating inhibitors for oxidative stress (N-acetylcysteine (NAC)) and apoptosis (Z-VAD-FMK). Accordingly, the oxidative-stress-related mechanisms were further assessed. StenB caused subG1 and G2/M accumulation but reduced the G1 phase in TNBC cells, while normal cells remained unchanged between the control and StenB treatments. The apoptosis behavior of TNBC cells was suppressed by StenB, whereas that of normal cells was not suppressed according to an annexin V assay. StenB-modulated apoptosis signaling, such as for caspases 3, 8, and 9, was more significantly activated in TNBC than in normal cells. StenB also caused oxidative stress in TNBC cells but not in normal cells according to a flow cytometry assay monitoring reactive oxygen species, mitochondrial superoxide, and their membrane potential. StenB induced greater DNA damage responses (γH2AX and 8-hydroxy-2-deoxyguanosine) in TNBC than in normal cells. All these StenB responses were alleviated by NAC pretreatment. Collectively, StenB modulated oxidative stress responses, leading to the antiproliferation of TNBC cells with little cytotoxicity in normal cells. [ABSTRACT FROM AUTHOR]

Ginger-derived compounds are abundant sources of anticancer natural products. However, the anticancer effects of (E)-3-hydroxy-1-(4′-hydroxy-3′,5′-dimethoxyphenyl)-tetradecan-6-en-5-one (3HDT) have not been examined. This study aims to assess the antiproliferation ability of 3HDT on triple-negative breast cancer (TNBC) cells. 3HDT showed dose-responsive antiproliferation for TNBC cells (HCC1937 and Hs578T). Moreover, 3HDT exerted higher antiproliferation and apoptosis on TNBC cells than on normal cells (H184B5F5/M10). By examining reactive oxygen species, mitochondrial membrane potential, and glutathione, we found that 3HDT provided higher inductions for oxidative stress in TNBC cells compared with normal cells. Antiproliferation, oxidative stress, antioxidant signaling, and apoptosis were recovered by N-acetylcysteine, indicating that 3HDT preferentially induced oxidative-stress-mediated antiproliferation in TNBC cells but not in normal cells. Moreover, by examining γH2A histone family member X (γH2AX) and 8-hydroxy-2-deoxyguanosine, we found that 3HDT provided higher inductions for DNA damage, which was also reverted by N-acetylcysteine. In conclusion, 3HDT is an effective anticancer drug with preferential antiproliferation, oxidative stress, apoptosis, and DNA damage effects on TNBC cells. [ABSTRACT FROM AUTHOR]

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect. [ABSTRACT FROM AUTHOR]

The anticancer effects and mechanisms of marine sponge Aaptos suberitoides were rarely assessed, especially for methanol extract of A. suberitoides (MEAS) to breast cancer cells. This study evaluated the differential suppression effects of proliferation by MEAS between breast cancer and normal cells. MEAS demonstrated more antiproliferation impact on breast cancer cells than normal cells, indicating oxidative stress-dependent preferential antiproliferation effects on breast cancer cells but not for normal cells. Several oxidative stress-associated responses were highly induced by MEAS in breast cancer cells but not normal cells, including the generations of cellular and mitochondrial oxidative stress as well as the depletion of mitochondrial membrane potential. MEAS downregulated cellular antioxidants such as glutathione, partly contributing to the upregulation of oxidative stress in breast cancer cells. This preferential oxidative stress generation is accompanied by more DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in breast cancer cells than in normal cells. N-acetylcysteine reverted these MEAS-triggered responses. In conclusion, MEAS is a potential natural product for treating breast cancer cells with the characteristics of preferential antiproliferation function without cytotoxicity to normal cells in vitro. [ABSTRACT FROM AUTHOR]

A novel nitrated [6,6,6]tricycles-derived compound containing nitro, methoxy, and ispropyloxy groups, namely SK1, was developed in our previous report. However, the anticancer effects of SK1 were not assessed. Moreover, SK1 contains two nitro groups (NO2) and one nitrogen-oxygen (N-O) bond exhibiting the potential for oxidative stress generation, but this was not examined. The present study aimed to evaluate the antiproliferation effects and oxidative stress and its associated responses between oral cancer and normal cells. Based on the MTS assay, SK1 demonstrated more antiproliferation ability in oral cancer cells than normal cells, reversed by N-acetylcysteine. This suggests that SK1 causes antiproliferation effects preferentially in an oxidative stress-dependent manner. The oxidative stress-associated responses were further validated, showing higher ROS/MitoSOX burst, MMP, and GSH depletion in oral cancer cells than in normal cells. Meanwhile, SK1 caused oxidative stress-causing apoptosis, such as caspases 3/8/9, and DNA damages, such as γH2AX and 8-OHdG, to a greater extent in oral cancer cells than in normal cells. Siilar to cell viability, these oxidative stress responses were partially diminished by NAC, indicating that SK1 promoted oxidative stress-dependent responses. In conclusion, SK1 exerts oxidative stress, apoptosis, and DNA damage to a greater extent to oral cancer cells than in normal cells. [ABSTRACT FROM AUTHOR]

Data regarding the effects of crude extract of Commelina plants in oral cancer treatment are scarce. This present study aimed to assess the proliferation-modulating effects of the Commelina sp. (MECO) methanol extract on oral cancer cells in culture, Ca9-22, and CAL 27. MECO suppressed viability to a greater extent in oral cancer cells than in normal cells. MECO also induced more annexin V, apoptosis, and caspase signaling for caspases 3/8/9 in oral cancer cells. The preferential antiproliferation and apoptosis were associated with cellular and mitochondrial oxidative stress in oral cancer cells. Moreover, MECO also preferentially induced DNA damage in oral cancer cells by elevating γH2AX and 8-hydroxyl-2′-deoxyguanosine. The oxidative stress scavengers N-acetylcysteine or MitoTEMPO reverted these preferential antiproliferation mechanisms. It can be concluded that MECO is a natural product with preferential antiproliferation effects and exhibits an oxidative stress-associated mechanism in oral cancer cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The antibiotic antimycin A (AMA) is commonly used as an inhibitor for the electron transport chain but its application in anticancer studies is rare. Recently, the repurposing use of AMA in antiproliferation of several cancer cell types has been reported. However, it is rarely investigated in oral cancer cells. The purpose of this study is to investigate the selective antiproliferation ability of AMA treatment on oral cancer cells. Cell viability, flow cytometry, and western blotting were applied to explore its possible anticancer mechanism in terms of both concentration‐ and exposure time‐effects. AMA shows the higher antiproliferation to two oral cancer CAL 27 and Ca9‐22 cell lines than normal oral HGF‐1 cell lines. Moreover, AMA induces the production of higher reactive oxygen species (ROS) levels and pan‐caspase activation in oral cancer CAL 27 and Ca9‐22 cells than in normal oral HGF‐1 cells, providing the possible mechanism for its selective antiproliferation effect of AMA. In addition to ROS, AMA induces mitochondrial superoxide (MitoSOX) generation and depletes mitochondrial membrane potential (MitoMP). This further supports the AMA‐induced oxidative stress changes in oral cancer CAL 27 and Ca9‐22 cells. AMA also shows high expressions of annexin V in CAL 27 and Ca9‐22 cells and cleaved forms of poly (ADP‐ribose) polymerase (PARP), caspase 9, and caspase 3 in CAL 27 cells, supporting the apoptosis‐inducing ability of AMA. Furthermore, AMA induces DNA damage (γH2AX and 8‐oxo‐2′‐deoxyguanosine [8‐oxodG]) in CAL 27 and Ca9‐22 cells. Notably, the AMA‐induced selective antiproliferation, oxidative stress, and DNA damage were partly prevented from N‐acetylcysteine (NAC) pretreatments. Taken together, AMA selectively kills oral cancer cells in an oxidative stress‐dependent mechanism involving apoptosis and DNA damage. [ABSTRACT FROM AUTHOR]

Nepenthes plants are a folk medicine in many Southeast Asia countries for curing diseases but its anticancer effect is rarely investigated. The objectives of this study were to investigate the antioral cancer ability of ethyl acetate extract of Nepenthes ventricosa x maxima (EANV). The preferential killing ability of EANV was determined by MTS-based cell viability assays. The bioactive effects were further screened by flow cytometry for apoptosis, oxidative stress, and DNA damage. At 24 h treatment, EANV dose dependently decreased six types of oral cancer cells, but the normal oral cells (HGF-1) kept a 90% viability. EANV also showed chronic antiproliferative effects and inhibited 3D sphere formation ability of oral cancer cells. Ca9-22 and CAL 27 oral cancer cells with high response to EANV increased subG1 populations and enhanced Annexin V- and pancaspase-detected apoptosis in these cells. EANV also induced the generation of reactive oxygen species (ROS) and mitochondrial superoxide and the dysfunction of mitochondrial membrane potential. Moreover, the oxidative DNA damage level such as 8-oxo-2′deoxyguanosine was increased in EANV-treated oral cancer cells. Taken together, EANV has a preferential killing effect against oral cancer cells associated with oxidative stress, apoptosis, and DNA damage, suggesting EANV as a potential antioral cancer agent. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: The genus Nepenthes of the pitcher plants contains several natural and hybrid species that are commonly used in herbal medicine in several countries, but its possible use in cancer applications remains unknown as yet. Methods: In this study, we investigated the antioral cancer properties using ethyl acetate extracts of the Nepenthes hybrid (Nepenthes ventricosa x sibuyanensis), namely EANS. The bioactivity was detected by a MTS-based cell proliferation assay and flow cytometric or Western blot analysis for apoptosis, oxidative stress, and DNA damage. Results: Treatment for 24 hrs of EANS inhibited all three types of oral cancer cells that were tested (Ca9-22, CAL 27, and SCC9), with just a small difference to normal oral cells (HGF-1). This antiproliferation was inhibited by pretreatments with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), and the apoptosis inhibitor (Z-VAD). EANS treatment increased the subG1 population and it also dose- and time-dependently induced annexin V- and pancaspase-detected apoptosis as well as cleaved caspases 3 and 9 overexpressions in the oral cancer cells (Ca9-22). After EANS treatment of Ca9-22 cells, intracellular ROS and mitochondrial superoxide (MitoSOX) were overexpressed and mitochondrial membrane potential (MMP) was disrupted. Moreover, DNA damages such as γH2AX and 8-oxo-2ʹ-deoxyguanosine (8-oxodG) were increased after EANS treatment to Ca9-22 cells. The EANS-induced effects (namely, oxidative stress, apoptosis, and DNA damage) were suppressed by ROS scavenger. Conclusion: Our findings demonstrated that EANS inhibits ROS-mediated proliferation against oral cancer cells. [ABSTRACT FROM AUTHOR]

Soft corals-derived natural product, sinularin, was antiproliferative against some cancers but its effect and detailed mechanism on oral cancer cells remain unclear. The subject of this study is to examine the antioral cancer effects and underlying detailed mechanisms in terms of cell viability, oxidative stress, cell cycle analysis, and apoptosis analyses. In MTS assay, sinularin dose-responsively decreased cell viability of three oral cancer cells (Ca9-22, HSC-3, and CAL 27) but only little damage to oral normal cells (HGF-1). This cell killing effect was rescued by the antioxidant N-acetylcysteine (NAC) pretreatment. Abnormal cell morphology and induction of reactive oxygen species (ROS) were found in sinularin-treated oral cancer Ca9-22 cells, however, NAC pretreatment also recovered these changes. Sinularin arrested the Ca9-22 cells at G2/M phase and dysregulated the G2/M regulatory proteins such as cdc2 and cyclin B1. Sinularin dose-responsively induced apoptosis on Ca9-22 cells in terms of flow cytometry (annexin V and pancaspase analyses) and western blotting (caspases 3, 8, 9) and poly (ADP-ribose) polymerase (PARP). These apoptotic changes of sinularin-treated Ca9-22 cells were rescued by NAC pretreatment. Taken together, sinularin induces oxidative stress-mediated antiproliferation, G2/M arrest, and apoptosis against oral cancer cells and may be a potential marine drug for antioral cancer therapy. [ABSTRACT FROM AUTHOR]

PurposeRadiation combined with natural products may improve the radiosensitivity of cancer cells. This study investigated the potential of a combined modality treatment with Ultraviolet C (UVC; wavelength range 200–280 nm) and our previously identified anti-oral cancer agent (methanolic extracts ofCryptocarya concinnaroots; MECCrt) in oral cancer cells. Materials and methodsThe mechanism of the possible synergy of UVC and MECCrt was explored in terms of cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), and DNA damage analyses. ResultsIn cell viability (%) at 24 h treatment, the low doses of UVC (14 J/m2) and MECCrt (10μg/ml) resulted in slight damage to human oral cancer Ca9-22 cells (83.2 and 80.4) but was less harmful to human oral normal HGF-1 cells (93.4 and 91.8, respectively). The combined treatment of UVC and MECCrt (UVC/MECCrt) had a lower viability (54.5%) than UVC or MECCrt alone in Ca9-22 cells but no showed significant change in HGF-1 cells. In Ca9-22 cells, the expression of flow cytometry-based apoptosis (sub-G1 phase, annexin V, and pancaspase assays) was significantly higher in UVC/MECCrt than in UVC or MECCrt alone (p< 0.0001). Using flow cytometry, intracellular ROS levels of UVC/MECCrt and MECCrt alone were higher than for UVC alone. MitoMP change and H2A histone family member X (γH2AX; H2AFX)-based DNA damage were synergistically inhibited and induced by MECCrt/UVC compared to its single treatment in Ca9-22 cells, respectively. ConclusionUVC plus MECCrt treatment had selective killing and synergistic anti-proliferative effects against oral cancer cells involving apoptosis, oxidative stress, and DNA damage. This combination therapy appears to have a great clinical potential against oral cancer cells. [ABSTRACT FROM PUBLISHER]

Apoptosis is a key mechanism for enhanced cellular radiosensitivity in radiation therapy. Studies suggest that Akt signaling may play a role in apoptosis and radioresistance. This study evaluates the possible modulating role of amiloride, an antihypertensive agent with a modulating effect to alternative splicing for regulating apoptosis, in the antiproliferative effects induced by ionizing radiation (IR) in glioblastoma multiforme (GBM) 8401 cells. Analysis of cell viability showed that amiloride treatment significantly inhibited cell proliferation in irradiated GBM8401 cells ( p<0.05) in a time-dependent manner, especially in cells treated with amiloride with IR post-treatment. In comparison with GBM8401 cells treated with amiloride alone, with GBM8401 cells treated with IR alone, and with human embryonic lung fibroblast control cells (HEL 299), GBM8401 cells treated with IR combined with amiloride showed increased overexpression of phosphorylated Akt, regardless of whether IR treatment was performed before or after amiloride administration. The alternative splicing pattern of apoptotic protease-activating factor-1 ( APAF1) in cells treated with amiloride alone, IR alone, and combined amiloride-IR treatments showed more consistent cell proliferation compared to that in other apoptosis-related genes such as baculoviral IAP repeat containing 5 ( BIRC5), Bcl-X, and homeodomain interacting protein kinase-3 ( HIPK3). In GBM8401 cells treated with amiloride with IR post-treatment, the ratio of prosurvival (-XL,-LC) to proapoptotic (-LN,-S) splice variants of APAF1 was lower than that seen in cells treated with amiloride with IR pretreatment, suggesting that proapoptotic splice variants of APAF1 ( APAF1-LN,-S) were higher in the glioblastoma cells treated with amiloride with IR post-treatment, as compared to glioblastoma cells and fibroblast control cells that had received other treatments. Together, these results suggest that amiloride modulates cell radiosensitivity involving the Akt phosphorylation and the alternative splicing of APAF1, especially for the cells treated with amiloride with IR post-treatment. Therefore, amiloride may improve the effectiveness of radiation therapy for GBMs. Treatment of irradiated GBM tumors with amiloride inhibited cell proliferation in a time-dependent manner. Drug treatment alone showed increased expression of pAkt. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ethyl acetate Nepenthes extract (EANT) from Nepenthes thorellii × (ventricosa × maxima) shows antiproliferation and apoptosis but not necrosis in breast cancer cells, but this has not been investigated in oral cancer cells. In the present study, EANT shows no cytotoxicity to normal oral cells but exhibits selective killing to six oral cancer cell lines. They were suppressed by pretreatment of the antioxidant inhibitor N-acetylcysteine (NAC), demonstrating that EANT-induced cell death was mediated by oxidative stress. Concerning high sensitivity to EANT, Ca9-22 and CAL 27 oral cancer cells were chosen for exploring detailed selective killing mechanisms. EANT triggers a mixture of necrosis and apoptosis as determined by annexin V/7-aminoactinmycin D analysis. Still, they show differential switches from necrosis at a low (10 μg/mL) concentration to apoptosis at high (25 μg/mL) concentration of EANT in oral cancer cells. NAC induces necrosis but suppresses annexin V-detected apoptosis in oral cancer cells. Necrostatin 1 (NEC1), a necroptosis inhibitor, moderately suppresses necrosis but induces apoptosis at 10 μg/mL EANT. In contrast, Z-VAD-FMK, a pancaspase inhibitor, slightly causes necrosis but suppresses apoptosis at 10 μg/mL EANT. Furthermore, the flow cytometry-detected pancaspase activity is dose-responsively increased but is suppressed by NAC and ZVAD, although not for NEC1 in oral cancer cells. EANT causes several oxidative stress events such as reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization. In response to oxidative stresses, the mRNA for antioxidant signaling, such as nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), heme oxygenase 1 (HMOX1), and thioredoxin (TXN), are overexpressed in oral cancer cells. Moreover, EANT also triggers DNA damage, as detected by γH2AX and 8-oxo-2′-deoxyguanosine adducts. The dependence of oxidative stress is validated by the evidence that NAC pretreatment reverts the changes of cellular and mitochondrial stress and DNA damage. Therefore, EANT exhibits antiproliferation involving an oxidative stress-dependent necrosis/apoptosis switch and DNA damage in oral cancer cells. [ABSTRACT FROM AUTHOR]

The anticancer effect of pomegranate polyphenolic extract POMx in oral cancer cells has rarely been explored, especially where its impact on mitochondrial functioning is concerned. Here, we attempt to evaluate the proliferation modulating function and mechanism of POMx against human oral cancer (Ca9-22, HSC-3, and OC-2) cells. POMx induced ATP depletion, subG1 accumulation, and annexin V/Western blotting-detected apoptosis in these three oral cancer cell lines but showed no toxicity to normal oral cell lines (HGF-1). POMx triggered mitochondrial membrane potential (MitoMP) disruption and mitochondrial superoxide (MitoSOX) generation associated with the differential downregulation of several antioxidant gene mRNA/protein expressions in oral cancer cells. POMx downregulated mitochondrial mass, mitochondrial DNA copy number, and mitochondrial biogenesis gene mRNA/protein expression in oral cancer cells. Moreover, POMx induced both PCR-based mitochondrial DNA damage and γH2AX-detected nuclear DNA damage in oral cancer cells. In conclusion, POMx provides antiproliferation and apoptosis of oral cancer cells through mechanisms of mitochondrial impairment. [ABSTRACT FROM AUTHOR]

Simple Summary: Combined treatments with low side effects enhance anticancer applications. This study focusses on validating the potential synergistic antiproliferation of the combined treatment of ultraviolet-C and the coral-derived compound sinularin (UVC/sinularin) in oral cancer cells. This study confirms that UVC/sinularin synergistically and selectively inhibits oral cancer cell proliferation with low cytotoxicity on normal oral cells. The mechanisms involve the enhanced cellular and mitochondrial oxidative stress that cause apoptosis, DNA damage, and mitochondrial dysfunction in oral cancer cells. Combined treatment is increasingly used to improve cancer therapy. Non-ionizing radiation ultraviolet-C (UVC) and sinularin, a coral Sinularia flexibilis-derived cembranolide, were separately reported to provide an antiproliferation function to some kinds of cancer cells. However, an antiproliferation function using the combined treatment of UVC/sinularin has not been investigated as yet. This study aimed to examine the combined antiproliferation function and explore the combination of UVC/sinularin in oral cancer cells compared to normal oral cells. Regarding cell viability, UVC/sinularin displays the synergistic and selective killing of two oral cancer cell lines, but remains non-effective for normal oral cell lines compared to treatments in terms of MTS and ATP assays. In tests using the flow cytometry, luminescence, and Western blotting methods, UVC/sinularin-treated oral cancer cells exhibited higher reactive oxygen species production, mitochondrial superoxide generation, mitochondrial membrane potential destruction, annexin V, pan-caspase, caspase 3/7, and cleaved-poly (ADP-ribose) polymerase expressions than that in normal oral cells. Accordingly, oxidative stress and apoptosis are highly induced in a combined UVC/sinularin treatment. Moreover, UVC/sinularin treatment provides higher G2/M arrest and γH2AX/8-hydroxyl-2′deoxyguanosine-detected DNA damages in oral cancer cells than in the separate treatments. A pretreatment can revert all of these changes of UVC/sinularin treatment with the antioxidant N-acetylcysteine. Taken together, UVC/sinularin acting upon oral cancer cells exhibits a synergistic and selective antiproliferation ability involving oxidative stress-dependent apoptosis and cellular DNA damage with low toxic side effects on normal oral cells. [ABSTRACT FROM AUTHOR]

Breast cancer expresses clinically heterogeneous characteristics and requires multipurpose drug development for curing the different tumor subtypes. Many withanolides have been isolated from Physalis species showing anticancer effects, but the anticancer function of physapruin A (PHA) has rarely been investigated. In this study, the anticancer properties of PHA in breast cancer cells were examined by concentration and time-course experiments. In terms of cellular ATP content, PHA inhibited the proliferation of three kinds of breast cancer cells: MCF7 (estrogen receptor (ER)+, progesterone receptor (PR)+/−, human epidermal growth factor receptor 2 (HER2)−), SKBR3 (ER−/PR−/HER2+), and MDA-MB-231 (triple-negative). Moreover, PHA induced G2/M arrest in MCF7 and MDA-MB-231 cells. In terms of flow cytometry, PHA induced the generation of reactive oxygen species (ROS), the generation of mitochondrial superoxide, mitochondrial membrane potential depletion, and γH2AX-detected DNA damage in breast cancer MCF7 and MDA-MB-231 cells, which were suppressed by the ROS inhibitor N-acetylcysteine (NAC). In terms of flow cytometry and Western blotting, PHA induced apoptotic expression (annexin V, and intrinsic and extrinsic apoptotic signaling), which was suppressed by NAC and an apoptosis inhibitor (Z-VAD-FMK), in breast cancer cells. Therefore, PHA is a potential anti-breast-cancer natural product that modulates the oxidative-stress response, cell-cycle disturbance, apoptosis, and γH2AX-detected DNA damage. [ABSTRACT FROM AUTHOR]

Some withanolides, particularly the family of steroidal lactones, show anticancer effects, but this is rarely reported for withanolide C (WHC)—especially anti-breast cancer effects. The subject of this study is to evaluate the ability of WHC to regulate the proliferation of breast cancer cells, using both time and concentration in treatment with WHC. In terms of ATP depletion, WHC induced more antiproliferation to three breast cancer cell lines, SKBR3, MCF7, and MDA-MB-231, than to normal breast M10 cell lines. SKBR3 and MCF7 cells showing higher sensitivity to WHC were used to explore the antiproliferation mechanism. Flow cytometric apoptosis analyses showed that subG1 phase and annexin V population were increased in breast cancer cells after WHC treatment. Western blotting showed that cleaved forms of the apoptotic proteins poly (ADP-ribose) polymerase (c-PARP) and cleaved caspase 3 (c-Cas 3) were increased in breast cancer cells. Flow cytometric oxidative stress analyses showed that WHC triggered reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX) production as well as glutathione depletion. In contrast, normal breast M10 cells showed lower levels of ROS and annexin V expression than breast cancer cells. Flow cytometric DNA damage analyses showed that WHC triggered γH2AX and 8-oxo-2′-deoxyguanosine (8-oxodG) expression in breast cancer cells. Moreover, N-acetylcysteine (NAC) pretreatment reverted oxidative stress-mediated ATP depletion, apoptosis, and DNA damage. Therefore, WHC kills breast cancer cells depending on oxidative stress-associated mechanisms. [ABSTRACT FROM AUTHOR]

Some lichens provide the resources of common traditional medicines and show anticancer effects. However, the anticancer effect of Usnproliea barbata (U. barbata) is rarely investigated, especially for oral cancer cells. The aim of this study was to investigate the cell killing function of methanol extracts of U. barbata (MEUB) against oral cancer cells. MEUB shows preferential killing against a number of oral cancer cell lines (Ca9-22, OECM-1, CAL 27, HSC3, and SCC9) but rarely affects normal oral cell lines (HGF-1). Ca9-22 and OECM-1 cells display the highest sensitivity to MEUB and were chosen for concentration effect and time course experiments to address its cytotoxic mechanisms. MEUB induces apoptosis of oral cancer cells in terms of the findings from flow cytometric assays and Western blotting, such as subG1 accumulation, annexin V detection, and pancaspase activation as well as poly (ADP-ribose) polymerase (PARP) cleavage. MEUB induces oxidative stress and DNA damage of oral cancer cells following flow cytometric assays, such as reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) production, mitochondrial membrane potential (MMP) depletion as well as overexpression of γH2AX and 8-oxo-2′deoxyguanosine (8-oxodG). All MEUB-induced changes in oral cancer cells were triggered by oxidative stress which was validated by pretreatment with antioxidant N-acetylcysteine (NAC). In conclusion, MEUB causes preferential killing of oral cancer cells and is associated with oxidative stress, apoptosis, and DNA damage. [ABSTRACT FROM AUTHOR]

Marine sponge-derived manoalide has a potent anti-inflammatory effect, but its potential application as an anti-cancer drug has not yet been extensively investigated. The purpose of this study is to evaluate the antiproliferative effects of manoalide on oral cancer cells. MTS assay at 24 h showed that manoalide inhibited the proliferation of six types of oral cancer cell lines (SCC9, HSC3, OC2, OECM-1, Ca9-22, and CAL 27) but did not affect the proliferation of normal oral cell line (human gingival fibroblasts (HGF-1)). Manoalide also inhibits the ATP production from 3D sphere formation of Ca9-22 and CAL 27 cells. Mechanically, manoalide induces subG1 accumulation in oral cancer cells. Manoalide also induces more annexin V expression in oral cancer Ca9-22 and CAL 27 cells than that of HGF-1 cells. Manoalide induces activation of caspase 3 (Cas 3), which is a hallmark of apoptosis in oral cancer cells, Ca9-22 and CAL 27. Inhibitors of Cas 8 and Cas 9 suppress manoalide-induced Cas 3 activation. Manoalide induces higher reactive oxygen species (ROS) productions in Ca9-22 and CAL 27 cells than in HGF-1 cells. This oxidative stress induction by manoalide is further supported by mitochondrial superoxide (MitoSOX) production and mitochondrial membrane potential (MitoMP) destruction in oral cancer cells. Subsequently, manoalide-induced oxidative stress leads to DNA damages, such as γH2AX and 8-oxo-2'-deoxyguanosine (8-oxodG), in oral cancer cells. Effects, such as enhanced antiproliferation, apoptosis, oxidative stress, and DNA damage, in manoalide-treated oral cancer cells were suppressed by inhibitors of oxidative stress or apoptosis, or both, such as N-acetylcysteine (NAC) and Z-VAD-FMK (Z-VAD). Moreover, mitochondria-targeted superoxide inhibitor MitoTEMPO suppresses manoalide-induced MitoSOX generation and γH2AX/8-oxodG DNA damages. This study validates the preferential antiproliferation effect of manoalide and explores the oxidative stress-dependent mechanisms in anti-oral cancer treatment. [ABSTRACT FROM AUTHOR]

The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231) cells, but showed less effect on breast normal (M10) cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD) flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose) polymerase (PARP), caspases 3, 8, and 9. These caspases and PARP activations were suppressed by N-acetylcysteine (NAC) pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG)). In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells. [ABSTRACT FROM AUTHOR]