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

Abstract

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.

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

“…Many factors such as oxidative stresses may induce apoptosis [ 33 ]. Because ROS generation following UVC [ 34 ], WFA [ 14 ], or UVC/natural product [ 35 ] treatments are fast, the ROS detection time of WFA/UVC was chosen as 12 h. Using DCFH-DA staining, the ROS generation was detected as oxidative stress by flow cytometry. According to an ROS assay for oral cancer Ca9-22 and HSC-3 cells (top and middle, Figure 5 A), 12-h UVC/WFA treatment induces higher ROS (+) (%) populations than UVC, WFA, and control treatments ( Figure 5 B).…”

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

“…A combined treatment with natural products may alleviate radio- and chemo-resistance or side effects in cancer therapy [ 5 , 6 ]. Recently, several potential UVC sensitizers for the antiproliferation of oral cancer cells were reported [ 13 , 15 , 16 , 17 ]. Accordingly, the current study evaluated the antiproliferation effect of the UVC/sinularin combined treatment of oral cancer cells.…”

“…For example, clinical agents such as cisplatin treatment (10 μM) and UVC irradiation (10 J/m 2 ) show synergistic suppression for proliferation to colorectal cancer cells [ 13 ]. Similarly, several kinds of combined treatments involving UVC and anticancer agents have been reported to provide the synergistic suppression of the proliferation of oral cancer cells, such as methanol extract of Cryptocarya concinna /UVC [ 16 ], ethyl acetate Nepenthes extract/UVC [ 17 ], and sulfonyl chromen-4-ones/UVC [ 15 ]. Therefore, the drug discovery of UVC radiosensitizers has the potential to improve oral cancer therapy.…”

“…For oral cancer cells, UVC irradiation doses ranging from 12 to 14 J/m 2 have a potential for ROS production [ 16 , 17 , 35 ] ( Figure 5 ), and sinularin at 23.5 [ 24 ], 2 and 3 μM ( Figure 5 ) also induces ROS production. Following the UVC/sinularin treatment, oral cancer cells generate several oxidative stress inductions, such as ROS production, MitoSOX generation, and MitoMP destruction.…”

“…UVC exposure shows translational potential to inhibit the proliferation of glioblastoma multiforme cancer cells [ 12 ]. The combined treatments of clinical drugs [ 13 , 14 ], chemical agents [ 15 ], or natural products [ 16 , 17 ] with UVC irradiation may improve the antiproliferation of several types of cancer cells. Moreover, UVC irradiation inhibits tumor growth in an animal model without apparent side effects [ 11 , 18 ].…”