Tumors tend to adapt to the microenvironmental changes when they are threatened by death. In clinical practice, some tumors remain in quiescent conditions due to hypoplasia of their supplying blood vessels. Meanwhile, some tumor tissues remain dystrophic since they cannot obtain enough nutrients from hypoplastic blood vessels. Besides, selectively starving cancer cells can also make tumor cells to be malnourished, which is a metabolic-based therapy for cancers with tiny side effects. Cancer-starving therapies, such as dietary modification, inhibition of tumor angiogenesis, and aspartic acid deficiency, can effectively decrease the incidence of spontaneous tumors and slow the growth of primary tumors.
ADI is a suitable gene to be targeted for cancer gene therapy. As a description of our preliminary work, cytosolic ADI expression displayed a higher apoptosis-inducing efficiency, tumor-targeting specificity, and oncolytic activity. In order to exclude the actions of adenovirus on cells, we just used a pcDNATM4/TO/myc-His vector as an ADI expression vector without replacing the pCMV promoter with a phTERT promoter. The rapid growth of tumors requires a tremendous supply of nutrients including arginine. Tumor cells exhibiting ASS gene deficiency such as endometrial cancer are more sensitive to arginine deprivation than normal cells. Based on the cancer tissue specificity of ASS expression, we used MRC5 (ASS+), PC3 (ASS-), and HepG2 (ASS-) cell lines to explore whether ADI had the same effect on different cancer cell lines. As illustrated in fig 1, ADI expressed in the cytosol eventually induced cellular apoptosis of PC3 and HepG2 cells.
ADI-PEG20 has been proved to induce cellular autophagy and caspase-independent apoptosis by exhausting the arginine in the peripheral microenvironment of tumors . Notwithstanding, it is unknown whether cytosolic ADI has the same anti-tumor mechanism. We aimed at understanding whether ADI has a unique anti-tumor mechanism in vivo. Consequently, we screened the protein factors that would interact with ADI using the yeast hybrid method. FTL was screened out as revealed in fig 2. Co-IP results confirmed the interaction between ADI and FTL in cells. Fluorescence co-localization demonstrated that the interaction happened in the cytoplasm.
Ferritin is considered as the major iron storage protein, which participates in the regulation of cellular iron homeostasis. Mitochondrial function also requires iron replenishment from cytoplasmic ferritin. Thus, inhibition of ferritin directly results in dysfunction of the mitochondrial electron transport chain. To exclude the effect of ADI’s enzymatic activity on cellular metabolism, the catalytic residues of ADI were mutated into alanine residues. Cysteine398, the catalytic residue of ADI, was mutated into alanine398. Since alamine398 as an inert residue has no nucleophilic catalytic capacity, the mutation (C398△A398) effectively terminated the enzymatic activity of ADI. As presented in fig 3, ADI△(C398△A398) still induced a small number of cell death in PC3 and HepG2 cells. Overexpression of FTL neutralized the apoptotic effects on these two cells. Based on these facts, we speculated that FTL overexpression constituted the part of cytosolic FTL that had lost its function due to interaction with ADI. That said, ADI△(C398△A398) needs 3 days to induce cancer cell death, while ADI only needs 2 days as pointed out in fig 2. It can be seen that cytosolic ADI△ just induces a limited level of apoptosis through interacting with cytosolic FTL. The interaction between ADI and FTL is not the main reason for mitochondrial damage. In addition, as represented in Fig. 1, high concentration of arginine in the culture medium counteracted the cell death caused by cytosolic ADI expression. This result further suggests that arginine deprivation in the cytosol is the predominant mechanism for cytosolic ADI suppressing the growth of cancer cells.
Collected pieces of evidence in research papers have proven that arginine deprivation in vitro exerts its anticancer effects on various tumors by inducing mitochondrial damage and autophagy[5, 6, 20, 21]. Additionally, arginine deprivation inhibits nitric oxide synthesis in cells[22, 23]. Thus, arginine deprivation cannot damage the mitochondria by increasing nitric oxide biosynthesis in cells. David K. Ann and Hsing-Jien Kung also reported that mitochondrial damage is the principal explanation for cancer cell apoptosis induced by ADI-PEG20. Our MPTP experiments also confirmed that cytosolic ADI led to serious mitochondrial damage as presented in fig 4c. However, the exact mechanism regarding the apoptosis pathway induced by mitochondrial damage during arginine deprivation in vivo is still not clear.
Next, we checked the expression of some protein factors associated to the mitochondrial apoptosis pathway. As demonstrated in fig 4a and 4b, 2 days of arginine deprivation in vivo increased the expression of p53 and p53AIP1 proteins in PC3 and HepG2 cells. Ectopic expression of the p53AIP1 protein induced down-regulation of the mitochondrial Δψm (transmembrane potential) and release of cytochrome c from the mitochondria by interacting and inhibiting Bcl-2 in the outer membrane of the mitochondria. Clearly, after two days of starvation, increase in the expression of the p53AIP1 protein activated p53-dependent apoptosis by interacting with the same upregulated expression of the p53 protein[11, 26]. Ultimately, cytochrome C was released from the mitochondria. Casp3 and Casp9 were activated as delineated in fig 4d and 4e. At the latest stage of arginine deprivation in cells (for 4 days), the PC3 and HepG2 cells seemed to enter the initiative apoptosis process, due to the fact that increasing expression of Noxa, PUMA, Bax and Bak proteins would further aggravate mitochondrial damage[27, 28] as shown in fig 4a and 4b.
We further knocked down the mRNA levels of p53 and p53AIP1 to verify their action during arginine deprivation in cells. As portrayed in fig 5a, 5b, 5d, and supplementary fig S5, the knockdown effectively reduced the apoptosis rates in PC3 and HepG2 cells. p53 knockdown displayed better effects in terms of apoptosis inhibition compared to the p53AIP1 knockdown. Mitochondrial damage was also prevented by p53 knockdown, due to the higher fluorescence intensity of living cells exhibited in fig 5c. Consequently, p53-dependent apoptosis pathway was the major pathway induced by cytosolic ADI.
It is worth mentioning that mitochondrial damage was not the only factor leading to cancer cell death during arginine deprivation in the cytosol. Cellular autophagy was also reported to be induced by ADI-PEG20. Autophagy, the process of cellular self-eating, is usually triggered by starvation or stress, which is capable to degrade long-lived proteins and organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, ribosomes and the nucleus[29, 30]. We also proved that autophagy was induced by cytosolic ADI. The pEGFP-LC3 and pcDNA4-ADI plasmids were co-transfected into cells. With the expression of ADI, more proteins were converted from LC3-I to LC3-II as laid out in figure 6b. Cytosolic GFP-LC3-I was conjugated to phosphatidylethanolamine to form GFP-LC3-II during autophagy. GFP-LC3-II was subsequently recruited into autophagosomal membranes during the formation of autophagosomes. As depicted in figure 6a, green GFP fluorescent particles presenting around the nucleus were autophagosomes in two cancer cells. Thus, with the expression of ADI, the autophagy induced by arginine starvation was indeed taking place in these cells.
Hsing-Jien Kung reported that arginine deprivation in vitro could lead to cancer cell chromatin autophagy. He equally stipulated that prolonged arginine deprivation would cause mitochondrial dysfunction and generation of ROS, eventually resulting in DNA damage and nuclear membrane remodeling. Excessive autophagy leads to a giant aggregate of autophagosomes/autolysosomes fusion in the late stage of arginine deprivation in vitro. Stephen Gregory disclosed that chromatophagy was necessary for the survival of chromosomal instability in (CIN) cells. Chromatophagy is activated to remove the defective mitochondria in response to DNA damage. However, we had an additional view of chromatophagy. We reckon that arginine deprivation mobilizes cells to utilize endogenous arginine storage. Nucleosomes, especially histone 3 (H3), contain abundant arginine residues. Consequently, the cells attempt to obtain arginine from chromatophagy to maintain basic physiology during arginine deprivation. As displayed in fig 6f, nucleus budding occurred in HepG2 and PC3 cells 96 hours after co-transfection of the pcDNA4-ADI and pEGFP-LC3 plasmids. Chromatin fragment (blue fluorescence) and H3 proteins (red fluorescence) were displayed to co-localize in autophagosomes (GFP green fluorescence). This showed that ADI expressed in the cytosol also induced chromatin autophagy. H3 proteins present in autophagosomes implied the utility of histones arginine.