Comparison between cancer types

Interestingly, both p62 (Figure 5.11) and LC3B-II (Figure 5.12) was significantly elevated in the B16 melanoma group. While LC3B-II levels might suggest an increase in autophagosomal pool size, the increase in p62 would imply a failure to degrade these vesicles. In other words, these results are suggestive of lysosomal dysfunction. It is tempting to speculate that a compromised autophagy apparatus might contribute to the low tolerance observed in mice bearing B16 tumours (Chapter 3). Indeed, lysosomal failure are is usually associated with an increase in cell death (Loos and Engelbrecht, 2009). However, this interpretation is not supported by the observation that caspase 3 cleavage was lower in the B16 group compared to the EO771 group (Figure 5.16), and that PARP cleavage was not significantly different between groups (Figure 5.15). Another context where LC3B-II is increased with p62 is during pathogen subversion of autophagy processes (van Niekerk et al., 2016): Many viruses, bacteria and protozoans inhibit lysosomal fusion with autophagic vesicles, since these pathogens make use of the vesicles to avoid immune detection. Although mice did not show any overt signs of infection, this possibility cannot be ruled out.

Prosurvival signaling (Akt phosphorylation) did not differ significantly: although Akt phosphorylation on Ser473 was significantly higher in the EO771 group (Figure 5.12), phosphorylation at Thr308 was not significantly elevated. Similarly, though PARP levels was significantly elevated in EO771 group, cleaved PARP was not (Figure 5.14), and possibly related to non-apoptotic function of these uncleaved proteins (Schwerk and Schulze-Osthoff,

B16 cancer cells also exhibited the lowest tolerance (Chapter 3), this observation would suggest that increased apoptosis might be reflective of increased liver pathology. In conclusion, the main finding of this study was that mice bearing B16 tumours might exhibit signs of dysfunctional autophagy, since p62 and LC3B-II where concomitantly elevated, suggesting that lysosomal function and degradation of vesicle content could not be executed.

5.9 Conclusion

Comparison between groups receiving DXR did not demonstrate an expected increase in autophagy: although LC3B-II was elevated in the tumour group compared to DXR, no other marker indicated an increase in autophagy. The elevated p-mTOR/mTOR ratio in the tumour group compared to DXR might be reflective of increased cell proliferation or protein synthesis rather than an inhibition of autophagy. This interpretation would describe the lower p- mTOR/mTOR ratio in mice receiving DXR compared to tumour group as a result of cellular stress (i.e. in mice receiving DXR, cells disengaging anabolic activities usually activated by suppressing mTOR activation). Yet, this interpretation is not corroborated by increased caspase 3 cleavage in the tumour group as it is not clear why cells expressing apoptotic markers, would activate mTOR signalling.

In contrast, there was suggestive evidence that mice bearing B16 tumours might exhibit signs of a compromised autophagic process. The increase in LC3B-II suggest an increase in vesicle formation, whereas the elevated p62 imply that vesicle content (and the p62 inside the vesicle) does not undergo degradation, thus suggesting an inability of lysosomal vesicles to fuse with autophagosomes. Regarding the various functions of autophagy (Figure 5.1), an inability to fully execute the entire autophagic process to completion could explain the lower tolerance in mice bearing B16 tumours.

Measuring autophagy with western blotting is challenging as it provides a ‘snap shot’ of a dynamic process. In this regard, transgenic mice models have been introduced to measure autophagic activity in vivo (Castillo et al., 2013). Also, only a few proteins playing an executioner role (i.e. not induction) was measured and it is therefrom not possible to point out the purpose of autophagy in these cells. As an example, autophagic machinery upregulated in response to nutritional stress (fasting) might be subsequently repurposed for other functions such as cell-autonomous defence against pathogens (van Niekerk et al., 2016). Thus, high basal

levels of autophagy in both control and DXR groups might be comparable, suggesting autophagy is not an important response to a DXR challenge. Yet, this does not preclude the possibility that autophagy was differentially implemented in pursuit of diverging needs (clearance of damaged mitochondria in DXR groups, versus mobilisation of energy rich substrate in control groups). Expanding the range of proteins investigated might highlight such differences. In this regard, a study was subsequently conducted making use of a proteomic analysis (Chapter 6) in order to resolve the ambiguous results obtained in this chapter.

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