Lung cancer is very prevalent worldwide, it is the leading cause of cancer related deaths in men and second in women; there are 1.3 million deaths occurring each year due to lung cancer (Ji et al. 304). Even with treatment, there is a less than 5 year survival rate (Ji et al. 305). Surgical resection, if possible, is followed by chemotherapy or radiotherapy; however, lung cancer cells have a resistance to these types of therapy which is why there is little efficacy with treatment (Ji et al. 305). Fighting cancer cells on a molecular level is the best method for reducing these statistics (Ji et al. 305). In the study Regulation of apoptosis and radiation sensitization in lung cancer cells via the SirT1/NF-ᴋB/Smac pathway, they focused on the radiosensitivity of lung cancer cells and basal molecular mechanisms associated with lung cancer, both in relation to Sirtuin 1 (SirT1) expression levels.
Two lung cancer cell lines from the non-small cell lung carcinoma (NSCLC) family were studied during this experiment: A549 cell lines associated with lung adenocarcinoma and H460 cell lines associated with large lung cell carcinoma (Ji et al. 312). These two types of lung cancer, along with squamous lung cell carcinoma, account for 75% of lung cancer cases (Ji et al. 312). It has been reported in other studies, as well as observed in this study, that A549 cells are more resistant to radiation while H460 cells are more susceptible to both radiation and apoptosis (Ji et al. 312). During this study, cell cultures were plated from both cell lines and were administered the same conditions for comparison (Ji et al. 305-307). One group of cells were pretreated with a siRNA (siSirT1) which is a knockdown regulator of SirT1; other cells were pretreated with resveratrol which activates expression of SirT1; and another group of cells were pretreated with BAY11-7082 (BAY) which inhibits the expression of nuclear factor-ᴋB (NF-ᴋB) (Ji et al. 305-307). Some of these cells from each treatment group were exposed to ionizing radiation while others were not ( Ji et al. 305-307). Control cells were neither exposed to radiation nor pretreated with anything (Ji et al. 305-307).
SirT1 is part of the mammalian silent information regulator family and is found in the cell nucleus; it is involved in regulating many molecular activities including: regulating cell survival and stress, inhibiting cell differentiation, regulating the cell cycle, inhibiting apoptosis, and tumorigenesis (Ji et al. 305). As a NAD+-dependent histone deacetylase, SirT1 is an enzyme with many substrates; it regulates both histones and non-histones, including NF-ᴋB (Ji et al. 305). Regulated by NF-ᴋB, Smac (second mitochondria-derived activator of caspases) is located in the intermembrane space of the mitochondria and is responsible for activating caspase-3 and caspase-9 (Ji et al. 305). Caspases are cysteine proteases that are important for regulation of apoptosis as well (Ji et al. 305). In leukemia, colon cancer, prostate cancer and lung cancer, SirT1 is overexpressed; this could be the reason why lung cancer cells have a resistance to both chemotherapy and radiotherapy (Ji et al. 311- 312). This study looked at SirT1/NF-ᴋB/Smac pathway to determine if modulating expression of any of these proteins could change the cells response to radiosensitivity, radiation-induced DNA damage, or apoptotic behavior (Ji et al. 305).
Results of this study suggest that SirT1 is a major player in radiosensitivity of lung cancer cell lines (Ji et al. 307). First, SirT1 expression levels were measured in the control groups to provide a baseline for the results; this showed that A549 cells had a much higher expression of SirT1 than H460 cells (Ji et al. 308). Cell colonies were looked at under a stereomicroscope after only radiation was administered, after only siSirT1 was administered, and after both radiation and siSirT1 were administered together; these same methods were done using resveratrol as the pre treatment (Ji et al. 308). The surviving colonies of both cell lines were significantly reduced after radiation when SirT1 expression was inhibited using siSirT1 (Ji et al. 308). Not only did inhibiting expression of SirT1 enhance radiosensitivity, but enhancing expression of SirT1 by resveratrol reduced radiosensitivity (Ji et al. 307). These results show that SirT1 is important for radiosensitivity because, when it was knocked down, the cells were no longer able to proliferate during radiation and when it was enhanced, both cell lines were able to survive radiation treatment. This is great news and is a great start to understanding the molecular mechanisms relating to cellular radiation resistance. Further research into this area could improve the understanding of how SirT1 changes the cells mechanisms.
Next, they looked at radiation-induced DNA damage in the form of double strand breaks (DSB) in both lung cancer cell lines (Ji et al. 307). They looked at DSBs because they cause the most damaging effects of ionizing radiation (Ji et al. 307). When a double strand DNA break occurs due to radiation, H2AX histones are phosphorylated (ɣH2AX) at several foci along the DNA strand; ɣH2AX-foci can appear along the DNA within several thousand base pairs of the DSB (Ji et al. 307). The phosphorylated H2AX histones were immunofluorescent stained to see if radiation-induced DNA damage was enhanced or inhibited when modulating SirT1 (Ji et al. 307). Control cells had 0% ɣH2AX-foci present, whereas radiation alone caused a 38.2% increase of ɣH2AX-foci (Ji et al. 307-308). When SirT1 expression was inhibited with siSirT1 and radiation was administered, the presence of ɣH2AX-foci increased by 35.1% (Ji et al. 308). When cells were pretreated with resveratrol and SirT1 expression was increased, the ɣH2AX-foci presence was significantly reduced in both cell lines (Ji et al. 308). Overall, these results are astounding and show that SirT1 expression is an important component in causing DNA damage after radiation treatment in these lung cancer cell lines. Since high levels of SirT1 expression is correlated with radiation resistance in these cell lines, it makes sense that DNA DSBs would be less prevalent in cells with high SirT1 expression.
Also in this study, they looked at results from lung cancer cell lines A549 and H460 to determine if regulation of SirT1 would have any effect on radiation-induced apoptosis by looking at overall apoptotic rates (Ji et al. 308). They did this by observing the expression levels of SirT1 and the effects SirT1 had on the expression levels of NF-ᴋB and Smac (Ji et al. 308-310). Smac triggers the activation of cleavage products of caspase-3 and caspase-9; NF-ᴋB is a SirT1 substrate associated with cell apoptosis that also mediates Smac expression, as mentioned previously (Ji et al. 308-310). First, they looked at A549 cells that were treated with radiation alone, these cells had an apoptotic rate of 8.9%; however, when they were pretreated with siSirT1 and then treated with radiation, the apoptotic rate was 26.7%, this is a substantial increase in apoptotic rates; furthermore, when treated with resveratrol and radiation, the rate decreased to approximately 5.7% (Ji et al. 308-309). When H460 cells were treated with radiation only, they had an apoptotic rate of 19.3%; when they were treated with siSirT1 and then treated with radiation, their apoptotic rate increased to 31.2%; and when treated with resveratrol and radiation, there apoptotic rate decreased to approximately 8.2% (Ji et al. 308-309). The variation in apoptotic rates of cells treated with radiation alone is most likely due to the fact that SirT1 expression levels are higher in A549 control cells versus H460 control cells (Ji et al. 309). Next, they looked at SirT1, NF-ᴋB, and Smac expression levels (Ji et al. 309-310). Smac and NF-ᴋB expression levels are higher in H460 control cells while SirT1 expression, as previously stated, is higher in A549 control cells (Ji et al. 309-310). When cells were pretreated with siSirT1, expression levels of SirT1 were reduced, as expected and has been shown in all previous areas of this research; NF-ᴋB and Smac expression levels were increased in both cell lines, but substantially in A549 cells, this is related to the fact that the control cells from this line had less expression of these proteins initially (Ji et al. 309-310). Cells pretreated with resveratrol showed increased levels of SirT1 expression, also expected; NF-ᴋB and Smac levels decreased in both cell lines (Ji et al. 309-310). They also pretreated cells from both cell lines with a NF-ᴋB inhibitor, BAY, and saw that apoptotic rates and Smac expression decreased in both cell lines (Ji et al. 310). Lastly, they looked at the activated cleavage caspase-3 and caspase-9 proteins which were barely seen, if seen at all, in control cells (Ji et al. 308). In A549 cells, radiation alone and resveratrol pretreatment increased the presence of the activated caspases by only trace amounts but pretreatment with siSirT1 showed an obvious increase in these proteins (Ji et al. 308). In H460 cells, radiation alone and siSirT1 pretreatment showed a substantial increase in these proteins while resveratrol pretreatment showed an obvious reduction in these activated caspases (Ji et al. 308). All of this data is in agreement and shows that expression levels of SirT1 is important for regulating expression of both NF-ᴋB and Smac. This research also shows that Smac expression and activated cleavage products of caspases are positively correlated with each other.
Overall, the results of this study made it obvious that SirT1 is associated with radiation sensitivity and apoptosis regulation in lung cancer cell lines A549 and H460. The results made it clear that downregulating SirT1 would reduce the resistance to radiation that the cells currently possess. If radiation resistance can be reduced by knockdown of SirT1, maybe the 5 year survival rate of lung cancer patients can be increased. This is at least a possibility for the outcomes of this research. Concurrently, the study showed that the SirT1/NF-ᴋB/Smac pathway is a major contributor to the responses that these specific lung cancer cells are having during and after radiation. This research is so important because the current methods of treating lung cancer are not efficient. In vivo studies still need to be done on this research to ensure realistic results for patients who are suffering from this deleterious disease, but this research is promising and could stimulate research for many other types of cancer that have similar responses to our current methods of treatment (Ji et al. 314).
The outline of this article was well organized and graphs made understanding the information much easier. I know that this research is credible because it is peer-reviewed with multiple sources cited for reference. The sources are relevant and timely to the research being done and the research is going to be critical for advancing treatment methods for cancers such as lung, prostate, colon, leukemia and many more. I believe the scientific community will have much success with further research on this pathway.
Work Cited
Ji, Kaihua, et al. “Regulation of Apoptosis and Radiation Sensitization in Lung Cancer Cells via the Sirt1/NF-ΚB/Smac Pathway.” Cellular Physiology and Biochemistry, vol. 48, no. 1, 16 July 2018, pp. 304–316., doi:10.1159/000491730.
