Arginine deimimase expressed in vivo activates the mitochondrial apoptosis pathway through inhibiting cytosolic ferritin and inducing chromotin autophagy

Background Based on its low toxicity, arginine starvation therapy has the potential to treat those malignant tumors that can’t be treated by surgery. Arginine deiminase (ADI) gene is indicated to be an idea cancer-suppressor gene. ADI expressed in vivo displays higher oncolytic efficiency than ADI-PEG20 (Pegylated Arginine Deiminase by PEG 20,000)[1]. However, it is still unknown whether cytosolic ADI has the same function mechanism as ADI-PEG20 or other underlying mechanisms in cells. The interaction of ADI and other protein factors was screened by yeast hybrid, and verified by co-immunoprecipitation and immunofluorescent staining. The effect of ADI inhibiting ferritin light-chain domain (FTL) on mitochondria damage was evaluated by site-directed mutation and flow cytometry. The apoptosis pathway of mitochondria control was analyzed by Western Blot and real-time PCR. The effect of p53 expression on cancer cell death was assessed by siTP53 transfection. The chromatin autophagy was explored by immunofluorescent staining and Western Blot.

excellent oncolytic efficiency [1]. Moreover, the promoter of human telomerase reverse transcriptase (hTERT) was utilized to control ADI expression in adenovirus, which ensured higher safety for normal cells. [1] However, it is still unknown about the underlying interaction mechanisms of ADI expressed in vivo, and the cellular response to rapid endogenous arginine deprivation. It will help to prevent side-effects when ADI gene is used for cancer gene therapy in future.
We found that cytosolic ADI can interact with FTL in cytoplasm. Cytosolic ferritin functions as storage and transport of iron ions in cells. We explored whether the interaction of ADI and FTL impairs mitochondria function through mutating the catalytic residue of ADI into alanine. Then, we detected the apoptosis pathway of mitochondria control. The increasing expression of p53 and p53AIP1 led to mitochondria damage at the early stage of arginine deprivation in vivo. At the later time of arginine deprivation in vivo, chromatin autophagy became worse and aggravated the mitochondria damage.
The molecular mechanism of cell death induced by ADI expressed in vivo is proved here to be associated with some cellular dysfunctions including ferritin inhibition, p53 expression upregulation, DNA damage and chromatin autophagy. The interaction between ADI and FTL is not the dominant reason of leading mitochondria damage. Arginine deprivation remains the key mechanism of ADI anticancer in the cytoplasm. In addition, cytoplasmic ADI will not interfere with other cellular function through interaction.

Plasmid construction
To construct pcDNA4-ADI, an ADI-overexpressing plasmid, ADI coding sequence was synthesized by Nanjing Genscript LTD and sub-cloned into the EcoR I/Xho I sites of pcDNA TM 4/TO/myc-His vector. C-myc tag was fused at the c-terminal of ADI protein. Two Total RNA was extracted from cells using Trizol (Invitrogen) following the manufacturer's instructions. The RNA concentration and purity were determined by spectrophotometry (NanoDrop Technologies Inc., LLC). One microgram of total RNA was used as the template for synthesizing complementary DNA (cDNA) by using the cDNA Synthesis Kit (Thermo Scientific). Quantitative RT-PCR (qRT-PCR) was performed by using SYBR Green PCR Master Mix with the StepOne Real-Time PCR System (Bio-Rad). 2 -△△Ct in relative quantification analysis method was used to calculate the change fold of mRNA among the different cells.
GAPDH was utilized as an internal control for the normalization. The primers used for RT-PCR were listed in supplementary tab S1.

Western Blot Analysis
Five micrograms of protein were electrophoresed in 10% SDS-PAGE gels and blotted to polyvinylidine difluoride membranes. Specific primary antibodies were detected with peroxidase-labeled secondary antibodies (ProteinTech Group Inc.) by using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical) per manufacturer's instructions.

Cancer cells apoptosis induced by ADI expressed in vivo.
ADI expressed in vivo will efficiently deplete intracellular arginine and lead to cell death.
Thus, we transfected pcDNA4-ADI plasmid into cancer cells to express ADI and assess apoptosis rate. Based on cancer tissue specificity of ASS expression

The interaction of ADI and FTL promoted the mitochondria damage.
To understand whether ADI has the unique anti-tumor mechanism in vivo, we screened

Cellular autophagy induced by ADI expressed in vivo.
Cellular autophagy was detected because nutrient starvation is the major reason to trigger excessive autophagy [12]. Assay for microtubule-associated protein 1A/1B-light chain 3 (LC3) is the basic protocol for the detection of autophagosome. A cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II) during autophaty, which is recruited to autophagosomal membranes.

Discussion
Tumors are very smart, which will actively adapt to the changes of microenvironments when they are threatened by death. In clinic, some tumors always stay in quiescent condition because of the hypoplasia of their blood vessels. Some tumor tissues remain in hunger since they can not obtain enough nutrient substances through hypoplastic blood vessels. What's more, selectively starving cancer cells can also make tumors keep in hunger, which is the metabolic-based therapy for cancers with tiny side effects. Cancerstarving therapies, such as dietary modification, tumor angiogenesis inhibition and aspartic acid deficiency, can effectively decrease the incidence of spontaneous tumors and slow the growth of primary tumors [14].
ADI was a good gene with the potential for cancer gene therapy. As the description of our preliminary work [1], ADI expressed in vivo was proved to possess higher apoptosisinducing efficiency, tumor-targeting specificity, and oncolytic activity [1]. In order to However, it is unknown whether ADI expressed in vivo has the same anti-tumor mechanism. We expect to understand whether ADI has the unique anti-tumor mechanism in vivo. Therefore, we screened the protein factors that might interact with ADI by yeast Autophagy, the process of cellular self-eating, is usually caused by starvation or stress, which is capable to degrade long-lived proteins and organelles such as endoplasmic reticulum, mitochondria, peroxisomes, ribosomes and nucleus [28,29]. We also verified that autophagy was aroused by ADI expressed in vivo as shown in fig 6a and 6b   The blots of PUMA were from the different parts of the same gel. The blots of p53AIP1 were from the different parts of the same gel. The blots of TP53 were from the different parts of the same gel. Other blots were from the different gels. Activity assay of Caspase 9 through caspase 9 assay kit (Colorimetric) (abcam. Ab65608). f/g: the relative quantification for protein expressions in PC3 and

Supplementary Files
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