Panoply™ Human MANF Knockdown Stable Cell Line

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced during endoplasmic reticulum (ER) stress responses induced by various stressors; however, its role in the radioresistance of tumor cells remains unclear. Here, researchers found that B16 melanoma cells release MANF following gamma-ray irradiation (2 Gy and 4 Gy), a process that is inhibited by the ER stress inhibitor 4-phenylbutyrate (4-PBA). While low-dose (1 Gy) gamma-ray irradiation did not induce MANF release, pre-treatment of 1 Gy-irradiated cells with recombinant MANF enhanced the DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the gamma-ray (2 Gy)-induced DNA damage response and p53 activation were suppressed, and reproductive cell death increased. Furthermore, MANF was found to activate the ERK signaling pathway. These findings suggest that MANF may serve as a novel target for overcoming radioresistance.

To determine whether MANF is involved in the DNA damage response, researchers evaluated the effect of MANF knockdown on the formation of γH2AX-53BP1 foci at DNA damage sites in B16 melanoma cells exposed to γ-irradiation. The results showed a significant reduction in foci formation (co-staining of γH2AX and 53BP1) in MANF-knockdown cells (Figure 1A). To examine long-term effects, they also assessed γH2AX-53BP1 foci formation 24 hours after γ-irradiation (4.0 Gy) as an indicator of unrepaired DNA damage. As shown in Figure 1B, significant DNA damage persisted in MANF-knockdown cells 24 hours post-irradiation. Given that p53 is a factor associated with radioresistance, the researchers further investigated p53 activation in MANF-knockdown cells. They found that p53 activation was significantly inhibited 1 hour after γ-irradiation in the knockdown cells, yet a persistent activated state was observed 24 hours post-irradiation (Figure 1C and D). These results strongly support the view that MANF participates in the DNA damage response and cell survival processes following γ-irradiation and contributes to conferring radioresistance to the cells.

Figure 1. Effect of MANF knockdown on radio resistance factor in B16 cells. (Tanaka Y, et al., 2020)