Combined Treatment with Low Cytotoxic Ethyl Acetate Nepenthes Extract and Ultraviolet-C Improves Antiproliferation to Oral Cancer Cells via Oxidative Stress

Ultraviolet-C (UVC) irradiation provides an alternative radiotherapy to X-ray. UVC sensitizer from natural products may improve radiotherapy at low cytotoxic side effects. The aim of this study is to assess the regulation for oral cancer cell proliferation by a combined treatment of UVC and our previously reported anti-oral cancer natural product (ethyl acetate extract of Nepenthes adrianii × clipeata; EANA). The detailed possible UVC sensitizing mechanisms of EANA such as effects on cell proliferation, cell cycle, apoptosis, and DNA damage are investigated individually and in combination using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay, flow cytometry, and western blotting at low dose conditions. In a 24 h MTS assay, the low dose EANA (5 μg/mL) and low dose UVC (12 J/m2) individually show 80% and combinedly 57% cell proliferation in oral cancer Ca9-22 cells; but no cytotoxicity to normal oral HGF-1 cells. Mechanistically, low dose EANA and low dose UVC individually induce apoptosis (subG1 accumulation, pancaspase activation, and caspases 3, 8, 9), oxidative stress (reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential depletion), and DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). Moreover, the combined treatment (UVC/EANA) synergistically induces these changes. Combined low dose treatment-induced antiproliferation, apoptosis, oxidative stress, and DNA damage were suppressed by the ROS scavenger N-acetylcysteine. In conclusion, UVC/EANA shows synergistic antiproliferation, oxidative stress, apoptosis, and DNA damage to oral cancer cells in an oxidative stress-dependent manner. With the selective killing properties of low dose EANA and low dose UVC, EANA provides a novel UVC sensitizing agent to improve the anti-oral cancer therapy.

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Oxidative Stress-Dependent Synergistic Antiproliferation, Apoptosis, and DNA Damage of Ultraviolet-C and Coral-Derived Sinularin Combined Treatment for Oral Cancer Cells

Cancers ◽

10.3390/cancers13102450 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2450

Author(s):

Sheng-Yao Peng ◽

Jen-Yang Tang ◽

Ruei-Nian Li ◽

Hurng-Wern Huang ◽

Chang-Yi Wu ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oral Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Combined Treatment ◽

Ultraviolet C ◽

Oral Cancer Cells ◽

Stress Dependent ◽

Oral 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.

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Low Dose Combined Treatment with Ultraviolet-C and Withaferin a Enhances Selective Killing of Oral Cancer Cells

Antioxidants ◽

10.3390/antiox9111120 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1120

Author(s):

Sheng-Yao Peng ◽

Yen-Yun Wang ◽

Ting-Hsun Lan ◽

Li-Ching Lin ◽

Shyng-Shiou F. Yuan ◽

...

Keyword(s):

Oral Cancer ◽

Cancer Cells ◽

Low Dose ◽

Combined Treatment ◽

Annexin V ◽

Withaferin A ◽

Single Treatment ◽

Flow Cytometric ◽

Ultraviolet C ◽

Oral Cancer Cells

Withaferin A (WFA), a Withania somnifera-derived triterpenoid, is an anticancer natural product. The anticancer effect of nonionizing radiation such as ultraviolet-C (UVC) as well as the combined treatment of UVC and WFA is rarely investigated. Low dose UVC and/or WFA treatments (12 J/m2 and/or 1 μM) were chosen to evaluate antioral cancer cell line effects by examining cytotoxicity, cell cycle disruption, apoptosis induction, and DNA damage. For two cancer cell lines (Ca9-22 and HSC-3), single treatment (UVC or WFA) showed about 80% viability, while a combined treatment of UVC/WFA showed about 40% viability. In contrast, there was noncytotoxicity to normal oral cell lines (HGF-1). Compared to single treatment and control, low dose UVC/WFA shows high inductions of apoptosis in terms of flow cytometric detections for subG1, annexin V, pancaspase changes as well as Western blotting for detecting cleaved poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) and luciferase assay for detecting Cas 3/7 activity. Low dose UVC/WFA also showed high inductions of oxidative stress and DNA damage in terms of flow cytometric detections of reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX) generation, and membrane potential (MitoMP) destruction, γH2AX and 8-oxo-2’deoxyguanosine (8-oxodG) types of DNA damages. For comparison, low dose UVC/WFA show rare inductions of annexin V, Cas 3/7 activity, ROS, MitoSOX, and MitoMP changes to normal oral HGF-1 cells. Therefore, low dose UVC/WFA provides a novel selectively killing mechanism to oral cancer cells, suggesting that WFA is a UVC sensitizer to inhibit the proliferation of oral cancer cells.

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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

International Journal of Molecular Sciences ◽

10.3390/ijms21176443 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6443 ◽

Cited By ~ 1

Author(s):

Sheng-Chieh Wang ◽

Yen-Yun Wang ◽

Li-Ching Lin ◽

Meng-Yang Chang ◽

Shyng-Shiou F. Yuan ◽

...

Keyword(s):

Oxidative Stress ◽

Flow Cytometry ◽

Dna Damage ◽

Oral Cancer ◽

Cell Viability ◽

Cancer Cells ◽

Untreated Control ◽

Combined Treatment ◽

Proliferation Inhibition ◽

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.

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Methanol Extract of Usnea barbata Induces Cell Killing, Apoptosis, and DNA Damage against Oral Cancer Cells through Oxidative Stress

Antioxidants ◽

10.3390/antiox9080694 ◽

2020 ◽

Vol 9 (8) ◽

pp. 694

Author(s):

Jen-Yang Tang ◽

Kuang-Han Wu ◽

Yen-Yun Wang ◽

Ammad Ahmad Farooqi ◽

Hurng-Wern Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oral Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Time Course ◽

Cell Killing ◽

Parp Cleavage ◽

Flow Cytometric ◽

Oral Cancer Cells

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.

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Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

Frontiers in Physiology ◽

10.3389/fphys.2017.00634 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 30

Author(s):

Hsueh-Wei Chang ◽

Ruei-Nian Li ◽

Hui-Ru Wang ◽

Jing-Ru Liu ◽

Jen-Yang Tang ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oral Cancer ◽

Cancer Cells ◽

Withaferin A ◽

Oral Cancer Cells

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

Pharmaceuticals ◽

10.3390/ph14100994 ◽

2021 ◽

Vol 14 (10) ◽

pp. 994

Author(s):

Kun-Han Yang ◽

Yu-Sheng Lin ◽

Sheng-Chieh Wang ◽

Min-Yu Lee ◽

Jen-Yang Tang ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Cell Growth ◽

Oral Cancer ◽

Growth Inhibition ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Soft Coral ◽

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.

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Manoalide Preferentially Provides Antiproliferation of Oral Cancer Cells by Oxidative Stress-Mediated Apoptosis and DNA Damage

Cancers ◽

10.3390/cancers11091303 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1303 ◽

Cited By ~ 8

Author(s):

Hui-Ru Wang ◽

Jen-Yang Tang ◽

Yen-Yun Wang ◽

Ammad Ahmad Farooqi ◽

Ching-Yu Yen ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oral Cancer ◽

Cancer Cells ◽

Annexin V ◽

Cancer Drug ◽

Dna Damages ◽

Atp Production ◽

Oral Cancer Cells ◽

Stress Dependent

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.

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