Prognostic significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in colorectal cancer treated with FOLFOX chemotherapy with or without aspirin

Abstract

Evasion of apoptosis is associated with treatment resistance and metastasis in colorectal cancer (CRC). Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic proteins (including p53 and PD-L1), anti-apoptotic proteins (BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2). The aim of this study was to determine the effect of folinic acid, 5-FU and oxaliplatin (FOLFOX) as a single agent and aspirin plus FOLFOX in various combinations on the aforementioned proteins in human CRC, SW480 cell line and rat models of N-Methyl-N-Nitrosourea (NMU)-induced CRC. In addition, effects of the NMU-induced CRC and chemotherapeutic regimens on haematological and biochemical parameters in the rat models were studied. Immunohistochemistry, immunofluorescence and immunoblot techniques were used to study the expression pattern of the related proteins in the human CRC cells pre- and post-treatment. Double contrast barium enema, post-mortem examination and histological analyses were used to confirm tumour growth and the effect of the treatment in vivo in rat models. Notably, we found in human mucinous CRC, a significant increase in expression of the BIRC7/Livin post-FOLFOX treatment compared with pre-treatment (p = 0.0001). This increase provides new insights into the prognostic role of BIRC7/Livin in evasion of apoptosis and facilitation of treatment resistance, local recurrence and metastasis particularly among mucinous CRCs post-FOLFOX chemotherapy. These poor prognostic features in the CRC may be further compounded by the significant suppression of DARC, PD-L1, PMS2 and overexpression of MSH2 and anti-apoptotic Bcl-2 and p53 proteins observed in our study (p < 0.05). Importantly, we found a significant reduction in expression of BIRC7/Livin and reactivation of DARC and PD-L1 with a surge in Annexin V expression in rat models of CRC cells post-treatment with a sequential dose of aspirin plus FOLFOX compared with other treatments in vivo (p <0.05). The mechanistic rational of these effects underscores the importance of expanded concept of possible aspirin combination therapy with FOLFOX sequentially in future CRC management. Validation of our findings through randomized clinical trials of aspirin plus FOLFOX sequentially in patients with CRC is therefore warranted.

Fig 1. BIRC7/Livin expression pattern and survival probability in African-American compared with White and Asian race.

A, Representative expression of BIRC7/Livin in CRC based on patient race. B, Representative effect of BIRC7/Livin expression level on CRC patient survival. C, Representative effect of BIRC7/Livin expression level and race on CRC patient survival.

Fig 2. Effect of aspirin treatment on BIRC7/Livin expression in DNA mismatch repair proficient, p53 mutant SW480 cell line in vitro.

A Is an Immunoblot showing effect of the 0.5 mM aspirin treatment on the SW480 compared to DMSO. PCNA was used as loading control. B, Quantitative analysis of BIRC7/Livin showing H score of the pattern of expression of the protein. C) SW480 cell line treated with 0.5 mM DMSO (vehicle control) and also with 0.5 mM aspirin for 24 hours (right). D) Describes the molecular pathway of aspirin metabolism, transport and target mechanises in colorectal cancer. This include inhibition of COX2, NF-kappaB and Wnt-B-Catenin signalling pathways for modulating BIRC7, DARC, PD-L1, Annexin V, PMS2, EGFR, BRAF, KRAS, PIK3CA, Bcl2, and p53 functions.

Fig 3. The expression pattern and functional significance of BIRC7/Livin pre and post- FOLFOX treatment in human colorectal cancer (scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

A) A bar chart showing the age distribution of different CRC subtypes used in the study in Zaria Nigeria. B) Normal colorectal sections showing negative expression of BIRC7/Livin Scale bar, 20 um. C) Colorectal mucinous adenocarcinoma sections showing positive expression of BIRC7/Livin pre-FOLOFOX treatment, Scale bar, 20 um. D) Colorectal mucinous adenocarcinoma lesions showing increase expression of BIRC7/Livin post-FOLFOX treatment. E-G) Colorectal cancer not otherwise specified (well and moderate differentiated) sections pre and post-FOLFOX chemotherapy showing increase expressions of BIRC7/Livin Scale bar, 20 um. H-K) BIRC7/Livin expression pattern typified in dot plot and showing pattern of the proteins expression using quantitative analysis technique. L-M) Representative IF images showing positive expression of BIRC7 in mucinous CRC histology and CRC not otherwise specified.

Fig 4. Double contrast barium enema and grossed appearance of the colon and rectum post and pre-treatment in rats.

A) Apparently normal colon lesion showing free uptake of the barium. B and C) Tumour involvement was evident by diffuse uptake of the barium at the anterior portion of the colon and little uptake at the mid to caudal portion. This signifies tumour involvement. D) Disarrayed uptake of the barium post-aspirin plus FOLFOX treatment. E, Grossed section of apparently normal colorectal lesions with arrows pointing to areas occluded by faecal materials. F) Tumour-bearing colorectal lesions Pre aspirin plus FOLFOX treatment with arrows showing areas with tumour involvement and total absent of faecal material. G) Tumour-bearing colorectal lesion post-treatment with aspirin plus FOLFOX in sequence showing apparently normal areas. H) Tumour-bearing colorectal lesion post-concurrent treatment with aspirin plus FOLFOX showing irregular distorted architecture of the colon and rectum with haemorrhagic areas.

Fig 5. The treatment effect of aspirin with or without FOLFOX on apoptotic cell death in colorectal cancer in vivo in rat (scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

A—F show various pattern of BIRC7/Livin expression in aspirin with or without FOLFOX treated rats and also in untreated group. G shows relative expression of the BIRC7 in rats typified in dot plot. H CRC-bearing rats treated with oxaliplatin showing obstructed colonic lumen devoid of faecal material. I show an enlarged abnormal Kidney (arrowed) from the oxaliplatin-treated rat observed during the post mortem. J Sections of enlarged kidney showing features of renal cell carcinoma with sarcomatoid differentiation with positive expression of PD-L1. K Sections showing different IF staining pattern upon treatment with aspirin only and aspirin plus FOLFOX in sequence and concurrent. L Sections of the liver showing preserved architecture that is devoid of haemorrhage in the group of rats treated with aspirin plus FOLFOX in sequence compared with concurrent treatment. M describes the possible pathways of the effect of sequential treatment of aspirin plus FOLFOX during tumour induction and chemotherapy. Several pathways are responsible for the aspirin and FOLFOX drug metabolism, transport and target mechanisms and they include but not limited to: COX2 inhibition, NFkB and Wnt-B-Catenin signalling pathways for aspirin; dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TYMP), thymidine kinase 1 (TK1) and uridine monophosphate synthetase (UMPS) for fluoropyrimidine component, 5-FU; and excision cross-complementing genes (ERCC) and glutathione S-transferases (GSTM) for the oxaliplatin (trans-1-diaminocyclohexane oxalateplatinum). These non-exhaustive have significant role in BIRC7, DARC, Annexin V, PD-L1, PMS2, EGFR, BRAF, KRAS, MDSC, PIK3CA, p53, BCL2 expression and functions. In NMU-induced colon carcinogenesis in rats, there is marked destruction of lymphocytes and granulocytes, significant reduction of red blood cells, platelet and total white blood cells. This was followed by reduction in alanine aminotransferase and alkaline phosphatase levels with increase in creatinine, electrolytes, urea, and aspartate aminotransferase levels. Sequential treatment of aspirin plus FOLFOX may confer a better anti-tumour immune response in CRC and this is justified by increase expression of PD-L, DARC and suppression of BIRC7.

Fig 6. Prognostic significance of p53, Bcl-2 and Annexin V in human colorectal cancer cells pre and post-treatment with FOLFOX n(scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

A) Colorectal mucinous adenocarcinoma sections showing positive expression of p53, Bcl-2 and Annexin V post-treatment with neoadjuvant FOLOFOX chemotherapy regimen, Scale bar, 20 um. B) Colorectal mucinous adenocarcinoma lesions showing increase in expression of Annexin V pre-FOLFOX treatment. C—D) Sections from colorectal cancer not otherwise specified (well differentiated and moderately differentiated types) post-FOLFOX chemotherapy showing strong positive expressions of p53, Bcl-2 and Annexin V. E-G) Quantitative analysis of p53, Bcl-2 and Annexin V showing the expression pattern typified in dot plot pre and post-treatment with FOLFOX.

Fig 7. Prognostic significance of p53, Bcl-2 and Annexin V in colorectal cancer cells post-treatment with FOLFOX with or without aspirin in rat (scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

A) Show positive expression of p53 and Bcl-2 and negative expression of Annexin V in normal colorectal sections. B to F), Show positive expression of p53, Bcl-2 and Annexin V in colorectal cancer sections pre and post-treatment with 25 mg/kg aspirin, aspirin plus FOLFOX in sequence, aspirin plus FOLFOX concurrent, and folinic acid treatment. G to I) Quantitative analysis of the p53, Bcl-2 and Annexin V from the rats showing expression pattern typified in dot plot post and pre-treatment with aspirin and aspirin plus FOLFOX in various combinations.

Fig 8. Prognostic significance of PD-L1, DARC and DNA mismatch repair proteins MSH2 and PMS2 in human CRC treated with and without neoadjuvant FOLOFOX (scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

Colorectal adenocarcinoma sections showing negative expression of the PD-L1, DARC and PMS2 and positive expression of MSH2 in mucinous CRC pre and post-FOLFOX treatment (A and B), and pre and post-FOLFOX treated CRC NOS (C—E).

Fig 9. Prognostic significance of PD-L1, DARC and DNA mismatch repair protein PMS2 in NMU-induced CRC in rat and treated with aspirin, and aspirin plus FOLOFOX in varous combinations (scale bar = 50 μm for H&E and scale bar = 100 μm for IHC).

A) Show negative expression of PD-L1, DARC and PMS2 in sections from non CRC-bearing rats. B) Show strong positive expression of PMS2 and negative expression of PD-L1 in the sections from the untreated CRC-bearing rats. C) CRC sections from the rat treated with 25 mg/kg aspirin showing negative expression of PD-L1 and mild expression of PMS2. D) Sections from aspirin plus FOLFOX sequential treated cells showing strongly positive expression of PD-L1 and PMS2 with mild expression of DARC. E) CRC sections treated with concurrent dose of aspirin plus FOLFOX showing mild expression of PD-L1 and DARC and strongly positive expression of PMS2. F) Sections treated with folinic acid showing focal expression of PD-L1, strong positive expression of DARC and mild expression of PMS2. G to I) Quantitative analysis of the PD-L1, DARC and PMS2 from the study showing expression pattern typified in dot plot post and pre-treatment with aspirin and aspirin plus FOLFOX in various combinations.