Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. It is currently thought that dysregulated cell cycle proliferation and apoptosis are important factors in the development of cancer . Therefore, it appears that exploiting the apoptotic potential of cancer cells might lead to new therapies that could be less toxic to normal cells due to the physiologically controlled survival pathway .
The ethanol extract of P. peruviana was reported to possess the lowest IC50 value against Hep G2 cells but had no cytotoxic effect on normal BALB/C mouse liver cells ; however, because these two cell lines are derived from different species, they are unable to provide strong evidence that the extract is a potential chemotherapeutic agent for lung cancer. Using cell lines of the same species, we found that the IC50 of 4βHWE at 24 h was 3.75 μg/mL for the HGF-1 (human normal gingival fibroblast)  cell line (data not shown) which was higher than the IC50 of the H1299 human lung cancer cell line (at 0.71 μg/mL; Figure 3). These results showed that H1299 cancer cells, but not a normal cell line, were sensitive to 4βHWE. However, we cannot exclude the possibility that there are tissue-specific differences between gingival and lung cells. Therefore, further in vitro cell cytotoxicity studies using a normal lung cell line, additional different lung cancer cell lines, and an in vivo nude mice study are necessary to prove that 4βHWE can inhibit tumor growth without major side effects.
Genomic lesions due to activation of the DNA damage response were detected, which then determined the cell fate, i.e., either cycle arrest or apoptosis (Figures 3, 4). However, both cell cycle interruption and apoptosis analyses are time-consuming and cannot be completed within a short time, e.g., at least 12~24 h of drug treatment is required. In contrast, the comet-NE assay was very sensitive to DNA strand breaks and can rapidly be performed , making it possible to determine the extent of DNA damage within only 2 h . Moreover, to confirm the comet assay results, we enhanced the dosage of 4βHWE according to the IC50 value; the results of treatment with the IC50 and other higher concentrations were similar, and exhibited a dose-responsive effect.
At high concentrations, the ethanol extract of P. peruviana induced cell cycle arrest, dose-dependent accumulation of the sub-G1 peak, and apoptosis through mitochondrial dysfunction . In this study, the main pure compound, 4βHWE from P. peruviana, was used to treat the H1299 lung cancer cell line. The results showed that cell cycle arrest at the G2/M phase (Figures 4, 5A), dose-dependent accumulation of a sub-G1 peak (Figure 4), and apoptosis were found in drug-treated cells (Figure 5). Furthermore, we found that cell cycle arrest at the G2/M phase was mediated by the ATM-NBS1 pathway which was activated by double strand breaks (data not shown).
The conventional alkaline comet assay is sensitive to DNA strand breaks, but is not sensitive to DNA lesions without strand breaks. In contrast, the NE in the comet-NE assay theoretically contains all of the repair enzymes and increases the incision of most kinds of DNA damage. Therefore, the comet-NE assay [34–36] is generally more sensitive than the traditional comet assay. However, different NEs from different cell lines may display differential excision activities for different types of DNA adducts. For example, the NE of NTUB1 cells is sensitive to ultraviolet C-induced DNA damage but is less sensitive to methyl methanesulfonate (MMS) than the NE of BFTC905 cells . Another problem with the comet-NE assay is that details of the types of lesions caused by the DNA damaging agent are not available. To resolve this problem, lesion-specific enzymes  can be used to replace the NE which may provide a more-specific and -sensitive way to detect DNA damage. Alternatively, immunodepletion coupled with the comet-NE assay might reveal the specific type of DNA damage .