The Role of Probiotics in Colorectal Cancer Management

Colorectal cancer (CRC) is one of the most common cancerous diseases worldwide and causes leading cancer-associated deaths. Several factors are related to the incidence of CRC such as unhealthy diet and lifestyle, heredity, metabolic disorders, and genetic factors. Even though several advanced medical procedures are available for CRC treatment, the survival rates are poor with many adverse treatments associated side effects, which affects the quality of life. Probiotics are a well-known bioactive candidate for the treatment of several diseases and ill-health conditions. The recent scientific evidence suggested that probiotic supplementation protects the CRC patients from treatment-associated adverse effects. The manuscript summarizes the influence of probiotic supplementation on the health status of CRC patients and discusses the possible mechanism behind the protective effect of probiotics against CRC. The literature survey revealed that beneficial impact of probiotic supplementation depends on several factors such as strain, dosage, duration of the intervention, host physiology, and other food supplements. The probiotic intervention improves the microbiota, releases antimicrobials and anticarcinogenic agents, helps to remove carcinogens, and improves the intestinal permeability, tight junction function, and enzyme activity in CRC patients. Besides, not all probiotic strains exhibit anti-CRC activities; it is necessary to screen the potent strain for the development of a probiotic-based therapeutic agent to control or prevent the incidence of CRC.


Introduction
Colorectal cancer (CRC) is one of the most common (∼1.4 million cases of CRC in 2012) cancerous disease worldwide and cause leading cancer-associated deaths (∼700 thousands of mortality) [1]. Several factors are associated with the incidence of CRC such as unhealthy diet and lifestyle, heredity, metabolic disorders, and genetic factors [2][3][4][5]. Indeed, 70% of the CRC incidents are related to environmental factors, and it has increased in technologically developed countries due to lack of physical activities [6,7]. e gut microbiota is closely associated with the incidence and development of CRC [8]. e altered gut microbiota can provoke the carcinogenesis by altering the immune response, epithelial hemostasis, metabolic profile and activity, DNA damage, and irregular cellular and molecular activities in colonocytes [8][9][10][11].
Even though several advanced medical procedures (chemotherapy, surgery, immune and radiation therapy) are available for CRC treatment, the survival rates are poor with many adverse treatment-associated side effects, which affects the quality of the life [8]. Probiotics are a well-known bioactive candidate for the treatment of several diseases, and ill-health conditions [12][13][14][15][16][17][18]. e administration of probiotics in an adequate amount confers the health benefits to the host by positive regulation of the gut microbiota. Dysregulation of the microbiota is one of the major factors of development of CRC. e studies suggested that the intervention of probiotics protects the CRC patients from treatment-associated adverse effects compared to the respective control populations in the studies [19][20][21]. e competition for adhesion site, production of microbicidal agents such as bacteriocin, improvement of intestinal permeability, release of bioactive metabolites, regulation of immune pathways, and stimulation of cell protective responses are the key functions of a potent probiotic strain, thereby aiding to prevent the tumorigenesis, not limited to, of CRC [8].
In this review, the authors discussed the influence of probiotic supplementation on the health status of CRC patients and highlighted the results of in vitro and in vivo studies related to CRC and probiotics. ey also discussed the possible molecular mechanism behind the health-promoting property of probiotics against CRC. e literature was collected from Scopus, PubMed, Google Scholar, and ResearchGate using the search terms "probiotics" and "colorectal cancer". e scientific documents (n � 50) were selected based on the information relevant to the scope of the current manuscript without any chronological restrictions.

Evidences of Anti-CRC Activities of Probiotics
2.1. In Vitro Studies. Baldwin et al. [22] demonstrated the effect of live or inactive probiotic strains (different concentrations of Lactobacillus acidophilus, and L. casei; total CFU are 1 × 10 6 , 1 × 10 7 , 1 × 10 8 , and 1 × 10 9 CFU per ml) on the apoptotic capacity of 5-fluorouracil  in the colorectal cancer cell line (LS513). e cotreatment of live or inactive L. acidophilus, L. casei (total probiotic concentration 1 × 10 8 CFU/ml), and 5-FU (100 µg/ml) enhanced the apoptotic efficiency (40%) of 5-FU in LS513 cells. e bacterial strains were inactivated through c irradiation or through microwave radiation. Irradiation-mediated inactivated probiotic strains also enhanced the apoptotic activity of 5-FU similar to the enhancing level of live probiotic strains at all concentrations. But microwave-treated probiotic strains reduced the apoptotic activity of 5-FU. Probiotic-mediated enhancement of apoptotic activity of 5-FU was dose-dependent. Probiotic strains (1 × 10 8 CFU per mL) and 5-FU exposure induced the caspase-3 activation and reduced the p21 expression faster in LS513 cells. e results suggested that use of potent probiotic strains can improve the efficacy of 5-FU [22]. Escamilla et al. [23] studied the effect of cell-free supernatants (CFS) from L. casei and L. rhamnosus GG on the invasion of human colorectal cancer cell line (HCT-116). CFS from both probiotic strains significantly reduced the HCT-116 cell invasion. CFS exposure reduced the matrix metalloproteinase-9 level and increased the zona occludens-1 level in HCT-116 cells. e inhibitory activities were not observed when HCT-116 cells were treated with CFS from commensal bacteria Bacteroides thetaiotaomicron. e active compounds were found to be present in the 50-100 kDa and >100 kDa fractions of CFS from both Lactobacillus strains. e study proved that secretory metabolites of L. casei and L. rhamnosus GG have anti-invasive activity in HCT-116 cells [23].
Orlando et al. [24] assessed the effect of live or heat-killed cells of L. paracasei IMPC2.1 and L. rhamnosus GG (10 8 CFU/mL) on the proliferation and apoptosis of gastric (HGC-27) and colon (DLD-1) cancer cell lines. Both live and heat-killed cells (L. paracasei IMPC2.1 and L. rhamnosus GG) effectively reduced the proliferation and induced the proapoptosis in both cancer cells in vitro. Hence, the cells of IMPC2.1 (heat-killed) can be used for the preparation probiotic-based functional food to improve the health status of CRC patients as a complementary regimen [24].
Soltan Dallal et al. [25] investigated the effect of CFS and bacterial extracts of probiotic strains (L. acidophilus ATCC 4356 and L. casei ATCC 39392) on the proliferation and apoptosis of colorectal cancer cell line (CaCo-2). Both CFS and bacterial extract of L. acidophilus ATCC 4356 and L. casei ATCC 39392 effectively reduced the proliferation, migration, and invasion of CaCo-2 cells and induced the apoptosis while cell necrosis was not induced by CFS treatment, whereas bacterial extract induced the cellular necrosis in CaCo-2 cells. e study suggested that CFS and bacterial extract of Lactobacillus strain impeded the malignant phenotype of CaCo-2 cells [25].
An and Ha [26] studied the effect of L. plantarum CFS on the characteristics of 5-FU-resistant HT-29 and HCT-116 cells. ey also examined the effect of L. plantarum CFS on the therapeutic capacity of 5-FU in 5-FU-resistant HT-29 and HCT-116 cells. Exposure (72 h) of L. plantarum CFS (10 μg) significantly reduced the expression of CD44, CD133, CD166, and ALDH1 in 5-FU-resistant HT-29 and HCT-116 cells. e combinational treatment of L. plantarum CFS (10 μg) and 5-FU (50 μM) hindered the Wnt/ β-catenin signaling and also increased the activity of caspase-3 and suppressed the formation and size of colonospheres in 5-FU-resistant HT-29 and HCT-116 cells. CFS from L. plantarum enhanced the therapeutic capacity of 5-FU in 5-FU-resistant colorectal cancer cells [26]. Cousin et al. [27] investigated the synergistic effect of Propionibacterium freudenreichii ITG P9 and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in HT-29 cells. Combination of Propionibacterium freudenreichii ITG P9 (CFS or metabolites) and TRAIL (TNF-related apoptosisinducing ligand) treatment synergistically induced proapoptosis and suppressed the antiapoptotic gene expression in HT-29 cells. e proapoptotic activity of combination therapy was dependent on death receptors TRAIL-R1/DR4, TRAIL-R2/DR5, and caspase activity. ITG P9-mediated fermented milk also exhibited the same apoptosis-inducing activity in combination with TRAIL indicating that CFS and metabolites of probiotics (ITG P9), and ITG P9-mediated fermented milk increased the efficacy of chemotherapy in CRC cells [27].
Kahouli et al. [29] demonstrated the effect of probiotic mix (L. acidophilus ATCC 314 and L. fermentum NCIMB 5221) on CaCo-2 cells. Probiotic mix treatment significantly reduced the proliferation of cancer cells and induced the apoptosis in CaCo-2 cells [29].
Saber et al. [30] examined the effect of secretion metabolites of Pichia kudriavzevii AS-12 on HT-29 and Caco-2 cells. Methanolic extract of secreted metabolites of Pichia kudriavzevii AS-12 (MEPK) was cytotoxic to HT-29 and Caco-2 cells, and the cytotoxic effect in HT-29 cells was comparable with that of 5-FU. e expression of BAD, caspase-3, caspase-8, caspase-9, and Fas-R was increased, while Bcl-2 expression was suppressed in MEPK-treated cancer cells. e level of proapoptotic genes (caspase-3, caspase-9, Fas-R in HT-29 cells, and Fas-R in Caco-2 cells) expression was higher in MEPK treated cells than 5-FU treated cells (positive control). Based on the results, the MEPK can be considered as a potent anticancer agent [30].
Sambrani et al. [31] studied the effect of Saccharomyces cerevisiae on the apoptosis, metastasis, and growth of HT-29 cells. CFS from S. cerevisiae exhibited antiproliferative activity in HT-29 cells by suppressing the expression of Bclxl and RelA and inducing the expression of PTEN and Caspas3 at 24 h posttreatment [31]. Gong et al. [32] evaluated the effect of L. acidophilus HB56003, Streptococcus thermophilus HB5621, Enterococcus faecalis HB62001, and Bifidobacterium longum HB55020 on human colonic smooth muscle strips. CFS, cellular fractions, and live cells of L. acidophilus HB56003, S. thermophilus HB5621, E. faecalis HB62001, B. longum HB55020 significantly inhibited the contractility of human colonic smooth muscle strips in vitro condition. NGnitro-L-arginine (an inhibitor of nitric oxide synthase) treatment reduced the inhibitory property of CFS from HB5621 and HB62001, but inhibitory property of HB55020 and HB56003 was not affected. e inhibitory activities of probiotics were dose-dependent [32] (Table 1).

In Vivo Studies.
Le Leu et al. [33] examined the effect of the intervention of probiotic (B. lactis; 1 × 10 11 CFU/g) or prebiotic (Hi-maize ® 958 or Hi-maize ® 260; high-amylose maize starch was used as a source of resistant starch 100 g/kg diet) or synbiotic (B. lactis + resistant starch) on incidence and development of colon neoplasm in azoxymethanemediated colonic neoplasm-induced Sprague Dawley rats. About 22 weeks of intervention showed that synbiotic preparation significantly reduced the incidence and proliferation of colon neoplasm. e changes in short-chain fatty acid content and variations in pH were also observed in the prebiotic group. Probiotic intervention exhibited no protective effects against CRC in the rat model. e study clearly revealed that the synbiotic intervention is a better protective agent compared to pre-or probiotic regimens [33].
Appleyard et al. [34] investigated the effect of probiotic (VSL#3) supplementation on colitis-associated CRC. e intervention of VSL#3 (a mixture of eight probiotic strains such as B. breve, B. infantis, B. longum, L. acidophilus, L. bulgaricus, L. casei, L. plantarum, and Streptococcus salivarius subsp. thermophilus) at the concentration of 5 × 10 9 CFU/100 g of body weight to trinitrobenzene sulfonic acid-mediated chronic colitis-induced Sprague Dawley rats significantly prevented the development of carcinoma, the incidence of high-grade dysplasia, and colon damages compared to control. e probiotic supplementation increased the expression of angiostatin, alkaline sphingomyelinase, and vitamin D receptor in experimental rats. e results suggested that VSL#3 protects the experimental rats from CRC development by diminishing the inflammatory responses and delay the progress of dysplasia [34]. Do et al. [35] studied the effect of probiotic (VSL#3) supplementation on colitis-associated CRC. VSL#3 (1.3 × 10 6 CFU/day) along with anti-inflammatory agent balsalazide (300 mg/kg body weight/day) effectively protected the experimental mouse from azoxymethane/dextran sodium sulfate-induced colitisassociated carcinogenesis. e supplementation of VSL#3 and balsalazide reduced the expression of p-STAT3 (phospho-signal transducer and activator of transcription 3) and BCL-2 (B-cell lymphoma 2) and decreased the level of MIP-1β (macrophage inflammatory protein 1 beta), MCP-1 (monocyte chemoattractant protein-1), IL-6 (interleukin-6), IL-10, and number of F4/80-positive macrophages and increased the BAX (BCL2-associated X protein) expression in CRC mice. e results suggested that the combination of VSL#3 and balsalazide could be an adjuvant therapeutic agent for CRC [35]. Another study revealed that the supplementation of VSL#3 was not interfering the azoxymethane-induced colitis-associated CRC development in Il10 -/mouse model but altered the mucosal-adherent microbiota [36].
e morphological changes were hindered in the DMH-mediated CRC-induced rats of probiotic-supplemented group compared to nonprobiotic group. e results suggested that L. rhamnosus GG and L. acidophilus exhibited better anti-CRC activities in DMH-mediated CRC-induced rats [37]. e further extended study revealed that the supplementation of synbiotic preparation consists of L. rhamnosus GG, L. acidophilus, and inulin displayed superior prophylactic activity by enhancing the antioxidant system in DMH-mediated CRC-induced rats compared to that of the supplementation of probiotic or prebiotic [38].
Mohania et al. [39] studied the effect of probiotics with or without piroxicam on DMH-induced colon carcinogenesis in male Wistar rats. e supplementation of L. acidophilus LaVK2 + B. bifidum BbVK3 and piroxicam significantly reduced the DMH-induced preneoplastic lesions (ACF, mucin-depleted foci) in rats. e ratio of aberrant crypts and ACF, large mucin-depleted foci, and proliferating cell nuclear antigen were also significantly decreased by probiotic supplementation.
e results suggested that supplementation of probiotics along with piroxicam exhibits better protective activity in DMH-mediated CRC-induced rats [39].

Evidence-Based Complementary and Alternative Medicine
Kumar et al. [40] investigated the effect of L. plantarum AS1 on DMH-induced colon carcinogenesis in male Wistar rats.
e pre-or post-or both (pre-and post-) supplementation of L. plantarum AS1 (10 9 CFU/day) for 5-21 weeks significantly improved the antioxidant status of DMH-induced CRC rat and positively altered the lipid peroxidation and selected biomarkers (superoxide dismutase, catalase, glutathione S-transferases, alkaline phosphatase, and acid phosphatase). e number and diameter of the tumor and the histopathological scores were reduced in AS1 supplemented group compared to control. e results suggested that AS1 supplementation, both preand postsupplementation, protects the DMH-induced CRC in the rat by enhancing the antioxidant system of the host [40].
Zhu et al. [41] investigated the effect of L. salivarius on DMH-induced colon carcinogenesis in male F344 rats. L. salivarius (5 × 10 8 or 1 × 10 10 CFU/Kg body weight/day for 15 weeks) supplementation improved the colonic microflora (reduced the Bacillus and Ruminococcaceae strains) and luminal metabolisms in DMH-mediated CRC-induced rats. e significant level of increase in short-chain fatty acids and a notable reduction in azoreductase activity was observed in the probiotic-treated group, while β-glucosidase and β-glucuronidase activities were not affected compared to control. e study suggested that the supplementation of L. salivarius positively altered the microbiota and enzyme activities in DMH-mediated CRC-induced rats [41].
Hu et al. [42] studied the effect of probiotic strains (L. plantarum or L. rhamnosus) in CT26 tumor-bearing BALB/c mice. BALB/c mice were presupplemented with L. plantarum or L. rhamnosus (1 × 10 9 CFU/day) for 14 days and CT26 carcinoma cells were introduced to induce cancer in mice.
e changes in immune regulations and status of tumor growth have been monitored in CT26 tumor-bearing mice. L. plantarum pre-exposure significantly reduced the Inhibited the contractility of colonic smooth muscle strips [32] MMP-9: matrix metalloproteinase-9; ZO-1: zona occludens-1; CFS: cell-free supernatant; 5-FU: 5-fluorouracil; TRAIL; TNF-related apoptosis-inducing ligand. 4 Evidence-Based Complementary and Alternative Medicine CT26 cell growth and increased the lifespan of tumorbearing mice by improving the 1-type CD4+ T differentiation, NK cell infiltration, CD8 + function, and IFN-c expression compared to L. rhamnosus pre-exposed group and control. e results proved that L. plantarum exhibited antitumor immune-enhancing property [42].
Zhang et al. [43] investigated the effect of L. salivarius Ren on DMH-induced colon carcinogenesis in male F344 rats. e supplementation of L. salivarius Ren (5 × 10 10 CFU/ Kg body weight/day) for 32 weeks reversed the DMH-induced altered microbiota in experimental rats. e level of Clostridiales, Bacteroides dorei, and Ruminococcus species have been reduced, and the amount of Prevotella species increased in L. salivarius Ren supplemented group. e results suggested that L. salivarius Ren supplementation protects the experimental animals from DMH-induced CRC via positive regulation of microbiota [43].
Gamallat et al. [44] investigated the effect of L. rhamnosus GG on DMH-induced colon carcinogenesis in Sprague Dawley rats. L. rhamnosus GG CGMCC 1.2134 (1 × 10 9 CFU/ day) intervention for 25 weeks significantly reduced the incidence, multiplicity, and volume of the tumor in DMHinduced CRC rat model. Also, the expression of TNF-α, COX-2, NFkB-p65, Bcl-2, and β-catenin were reduced, and Bax, p53, and casp3 expressions were increased in the probioticsupplemented group compared to the nonprobiotic group. e results indicated that L. rhamnosus GG CGMCC 1.2134 could diminish the CRC-associated inflammatory reactions, thereby protecting the host system [44].
Lenoir et al. [45] investigated the effect of L. casei BL23 on DMH-induced colon carcinogenesis in C57BL/6 mice. e C57BL/6 mice were presupplemented with 10 µl (1 × 10 8 CFU/µl) of L. casei BL23 on day (0, 14, and 28), and then CRC induction was started on the 35 th day and continued weekly during 10 weeks. Presupplementation of L. casei BL23 significantly protected the mice from CRC via altering the regulation of T reg and 17 T-cell-associated cytokines. Particularly, L. casei BL23 supplementation reduced the incidence of the tumor and the number of multiple plaque lesions and improved the histopathological score. e expression of IL-6, IL-10, IL-17, and TGF-β and the ratio of IL-10/TNF-α were increased in the probiotictreated group. Collectively, the results suggested that L. casei BL23 protects the mice from DMH-induced CRC via T reg and 17 T-cell regulation [45].
Mi et al. [46] assessed the chemoprotective effect and anti-CRC property of B. infantis in DMH and SW480 cell induced CRC rat model. e CRC induced animals were supplemented with B. infantis (1 × 10 9 CFU/day) and/or 5-FU + Oxaliplatin for 11 days, and the animals were examined for several pathological assessments. e level of IL-6, IL-1β, TNF-α levels, and 17 and 1 cells-associated cytokines were reduced and the level of CD4 + , CD25 + , Foxp 3+ , and T regs were increased in probiotic-supplemented CRC rat. e results collectively showed that the supplementation of probiotic effectively reduced the chemotherapy-associated health damages in a rat model [46].
Kahouli et al. [29] demonstrated the effect of probiotic mix (L. acidophilus ATCC 314 and L. fermentum NCIMB 5221) on Apc Min/+ CRC mouse model. e supplementation of probiotic formula consists of 0.5 × 10 10 CFU of L. acidophilus ATCC 314, and 0.5 × 10 10 CFU of L. fermentum per day for 12 weeks reduced the severity of CRC in Apc Min/+ CRC mouse model. e number and multiplicity of the tumor and expression of cellular proliferation markers were reduced significantly in the probiotic-treated group compared to control. e results claimed that the prepared probiotic regimen could be used as a biotherapeutic agent to avert the CRC [29].
Song et al. [47] studied the effect of probiotic Bifico on azoxymethane/dextran sodium sulfate-induced colitis-associated carcinogenesis in C57BL/6 mice. e supplementation of probiotic mix (B. longum, L. acidophilus, and E. faecalis; 1.2 × 10 7 CFU/day for 2 weeks pretreatment and continued till the end of the experiment) significantly reduced the tumor formation and intestinal inflammation, and improved the diversity and abundance of microbiota and altered the expression of CXCR2 ligand genes in azoxymethane/dextran sodium sulfate-induced colitis-associated cancer mice model [47].
Heydarii et al. [48] investigated the influence of supplementation of probiotics on the microRNA regulation in azoxymethane-induced CRC mice. About 5-month intervention of L. acidophilus (1 × 10 9 CFU/day) and B. bifidum (1 × 10 9 CFU/day) improved the expression pattern of miRNA-associated with cancer prevention. e relative expression of miR-135b, miR-155, and KRAS have been reduced while the expression of miR-26b, miR-18a, APC, PU.1, and PTEN were increased in the probiotic-treated group compared to nonprobiotic group. e study suggested that the probiotic exhibited antitumor property by regulating the miRNAs and associated genes in CRC experimental mice [48].
Lin et al. [49] revealed that the supplementation of L. acidophilus LA5 and/or B. animalis subsp. lactis BB-12 (5 × 10 7 CFU of single probiotic strain/day or 2.5 × 10 7 CFU each strain/day) and germinated brown rice extract (GBR; 10% in diet) for 10 weeks reduced the mucin-depleted foci formation, ACF-producing sialomucin and expression of anti-apoptotic Bcl-2 in azoxymethane/dextran sodium sulfate-induced CRC rat model. e supplementation of probiotics and GBR protects the CRC rat by increasing the expression of p53, Bax, caspase-3, and Bax/Bcl-2 ratio and restored the SOD activity. e results suggested that GBR and probiotic supplementation improved the antioxidant machinery of the host system and induced the apoptosis in tumor cells [49].
Sharaf et al. [50] studied the protective effect of probiotic strains (L. rhamnosus GG MTCC #1408 and/or L. acidophilus NCDC #15) along with anti-inflammatory drug (celecoxib) in DMH-induced CRC rats. Supplementation of 1 × 10 9 CFU/day of probiotics (single strain or multistrain) and/or celecoxib (6 mg/kg body weight) for 18 weeks significantly reduced the tumor burden and multiplicity of the tumor. e expression studies suggested that studied regimen effectively reduced the expression of antiapoptotic genes (Bcl-2, K-ras) and increased the tumor suppressor and proapoptotic genes (p53 and Bax) in the CRC rat model. e Evidence-Based Complementary and Alternative Medicine study suggested that the combination of multistrain probiotic preparation and celecoxib exhibited superior protective activity compared to single-strain regimen [50]. Another study showed that even 6 weeks of the intervention of probiotics, especially L. rhamnosus GG, and celecoxib significantly reduced the β-catenin, COX-2, and NF-κB expression, and formation of ACF in DMH-induced CRC rats [51] (Table 2).

Clinical Trials
Osterlund et al. [52] conducted a randomized, phase III, open-label, 2 × 3 factorial design study to investigate the influence of probiotic supplementation on chemotherapyinduced diarrhea in CRC patients (individuals with Dukes' stage B CRC or Dukes' stage C CRC or Dukes' stage D CRC who have undergone surgery). e patients received 5-FUbased postoperative adjuvant chemotherapy (at Helsinki University Central Hospital, Finland) for 24 weeks, and they were supplemented with probiotic strain L. rhamnosus GG (1-2 × 10 10 /day) capsules and guar gum fiber containing nutritional supplement during the adjuvant chemotherapy. e probiotic supplementation significantly reduced the frequency of diarrhea and abdominal discomfort. e results suggested that L. rhamnosus GG can be used as an adjuvant therapy to diminish chemotherapy-associated diarrhea and gastrointestinal discomfort [52]. Golkhalkhali et al. [53] conducted a double blind, randomized controlled trial with CRC patients to study the effect of strain-specific probiotic mix and ω-3 fatty acid on XELOX chemotherapy (at University of Malaya Medical Centre, Malaysia). e supplementation of probiotic preparation (a mixture of L. casei, L. acidophilus, L. lactis, B. bifidum, B. longum, and B. infantis strains; 30 × 10 9 CFU/sachet; 2 sachets per day for 4 weeks), and ω-3 fatty acid (2 g per day for 8 weeks) improved the health condition in CRC patients. e chemotherapy-associated inflammatory reactions were significantly nullified in the treatment group supplemented with probiotic and ω-3 fatty acid. e level of IL-6 was reduced in the treatment group compared to the placebo group. e study suggested that probiotic intervention reduced the chemotherapy-induced inflammatory dysregulation and improved the quality of life in CRC patients [53].
Rafter et al. [54] studied the effect of dietary synbiotic on the risk of CRC in polypectomized (n � 43) and CRC (n � 37; 6 individuals with Dukes' stage A CRC, 17 individuals with Dukes' stage B CRC, and 14 individuals with Dukes' stage C CRC) patients. e supplementation of synbiotic formula (L. rhamnosus GG, B. lactis Bb12, and oligofructose-enriched inulin) significantly reduced tumor proliferation and improved the barrier function in polypectomized patients. Moreover, the intervention of synbiotic formula regulated the microbiota, a notable level of increase in Lactobacillus and Bifidobacterium species, and a decrease in Clostridium perfringens have been observed in the fecal samples of the patients. e release of IFN-c and IL-2 were also altered after synbiotic supplementation in CRC patients. e results suggested that the supplementation of synbiotic preparation beneficially altered the CRC-associated biomarkers [54].
Gao et al. [55] investigated the effect of probiotic mix on CRC patients (individuals with CRC who have undergone radial colorectomy at Sixth People's Hospital affiliated Shanghai Jiao Tong University, Shanghai, China). e intervention of probiotic regimen (B. longum, L. acidophilus, and E. faecalis; 6 × 10 7 CFU/day) for five days significantly improved diversity and density of mucosa-associated microbiota and decreased the Fusobacterium species in CRC patients. e results suggested that probiotic can improve the health status of CRC patients via positive regulation of mucosal-associate microbiota [55].
Ishikawa et al. [56] studied the protective effect of dietary fiber and probiotics by conducting a randomized clinical trial with the human volunteers (who had colorectal tumors removal surgery). e volunteers were supplemented with L. casei strain Shirota (n � 96) or wheat bran (n � 95) or both (n � 96), and volunteers with no treatment (n � 93) for four years. e probiotic-supplemented group showed a significant reduction in the incidence of tumor formation compared to wheat bran supplemented group and control group after four years. Atypia of colorectal tumors has been significantly prevented by the probiotic treatment [56].
Worthley et al. [57] conducted a placebo-controlled double blind crossover trial to study the effect of supplementation of prebiotic, probiotic, and synbiotic preparation on the biomarkers of CRC in healthy human volunteers. All the volunteers were supplemented with probiotics (B. lactis; 1 × 10 9 CFU/g; 5 g/day), prebiotics (resistant starch; 25 g/ day), and synbiotics in a sequential way, and each intervention lasts for 4 weeks with no washout period. Changes in the microbiota, DNA methylation, epithelial proliferation, and biomarkers of CRC has been assessed after 4 weeks of each intervention. e results suggested that the supplementation of synbiotic preparation effectively altered the microbiota than other interventions. Moreover, synbiotic supplementation did not significantly affect the serum, fecal, and epithelial biomarkers [57].
Gianotti et al. [58] conducted a double-blind randomized controlled trial to study the effect of probiotics (B. longum and L. johnsonii; low dose 2 × 10 7 CFU per day or high dose 2 × 10 9 CFU per day) when supplemented perioperative to CRC patients (individuals with CRC who are undergoing elective colorectal surgery). e perioperative (pre-, on day, and postsurgery) supplementation of probiotics (high dose of B. longum and L. johnsonii) to CRC patients significantly reduced the members of Enterobacteriaceae in fecal samples. L. johnsonii was observed in the stool samples or in biopsy samples of CRC patients supplemented with probiotics. But presence of B. longum was not observed in the stool samples or in biopsy samples of CRC patients in probiotic group. Adherence of L. johnsonii was directly correlated with the probiotic dose. e expression of CD3, CD4, CD8, and lymphocyte subsets was increased, and the dendritic cells were not affected and activated, significantly. All the observed changes were directly correlated with the dose of the probiotic supplementation.
e study suggested that L. johnsonii can improve the health status of CRC patients by adhering the colonic mucosa and altering the microbiota and immune    Evidence-Based Complementary and Alternative Medicine diarrhea and infections and improved the transepithelial resistance and expression of tight junction protein in CRC patients [59]. Liu et al. [60] investigated the effect of perioperative probiotic supplementation on the serum zonulin level and postsurgery-related infectious complication in CRC patients (individuals with Dukes' stage A CRC, Dukes' stage B CRC, or Dukes' stage C CRC who are undergoing colorectal surgery). e supplementation of same probiotic formula [59] significantly reduced the serum zonulin level, duration of the postoperative antibiotic treatment, pyrexia, and infection in CRC patients. e p38 mitogen-activated protein kinase pathway was also hindered during probiotic supplementation. e results suggested that the probiotic formulation comprises L. plantarum, L. acidophilus, and B. longum improved the serum zonulin level and postsurgeryrelated infectious complications in CRC patients [60]. Hibberd et al. [61] studied the effect of probiotics on the microbiota in CRC patients (individuals with CRC stage I-III).
e CRC patients (n � 8) were supplemented with probiotics (ProBion Clinica, 2 tablets containing 1.4 × 10 10 CFU of B. lactis, 7 × 10 9 CFU of L. acidophilus, and 630 mg inulin per day) for 8-78 days until the day of surgery (intervention duration varied depends on the duration between the diagnosis to surgery period). e biopsy samples (both tumor and normal mucosa) were collected from CRC patients of probiotics group, and CRC patients (n � 7) of nonprobiotic group during both colonoscopy and surgery. Normal mucosal biopsies were also collected from noncancer control groups (n � 21; individuals with normal colonic mucosa) during colonoscopy. Fecal samples were obtained from all participants (CRC patients and noncancer individuals) after colonoscopy and from CRC patients at surgery. e results showed that the microbiota of tumorassociated samples was enriched with tumor-related microbial niche compared to control subjects. Probiotic intervention improved the diversity and abundance of butyrate-producing bacteria (Clostridiales and Faecalibacterium species) in the fecal and mucosal (tumor and normal mucosa) microbiota of CRC patients. Probiotic intervention also reduced the level of Fusobacterium and Peptostreptococcus species (which are considered as tumorinducing microbial agents) in fecal microbiota of CRC patients [61].
Lee et al. [62] investigated the effect of probiotic (L. rhamnosus R0011 and L. acidophilus R0052; 2 × 10 9 CFU/ tablet, twice a day for 12 weeks) on the quality of life in CRC survivors (individuals who have completed the treatment between 6 weeks and 2 years before the study) by conducting a randomized, double-blind placebo-controlled trial. e quality of life improvement was assessed by questionnaires. e results suggested that the supplementation of probiotic formula improved the health-span (improvement in irritable bowel symptoms, CRC-related health issues, functional wellbeing scores) of the participants significantly [62].
Aisu et al. [63] studied the effect of perioperative probiotic supplementation on the postsurgery-related infectious complication in CRC patients (individuals with CRC stages I, II, IIIA, IIIB, and IV who are undergoing elective colorectal surgery). e supplementation of BIO-THREE ® (Enterococcus faecalis T110, Clostridium butyricum TO-A, and Bacillus mesentericus TO-A) to perioperative CRC patients for 3-15 days (before surgery) significantly reduced the postoperational superficial incisional surgical site infections compared to nonprobiotic group and also improved the microbiota and immune system positively [63]. Tan et al. [64] examined the effect of perioperative probiotic (HEXBIO ® ) supplementation in promoting the recovery and returning to normal gut function in CRC patients (individuals with CRC stages I, II, III, and IV who are undergoing elective colorectal surgery). e perioperative supplementation of HEXBIO ® (a mixture of L. acidophilus, L. lactis, B. infantis. B. bifidum, and B. longum; 30 × 10 9 CFU/sachet; twice per day) to CRC patients for seven days (prior to surgery) significantly reduced the time required for regaining normal gut function after surgery and also reduced the duration of the hospital stay compared to the placebo group.
e study suggested that the perioperative supplementation probiotic formulations could help to improve the health status of CRC patients after surgery [64]. Yang et al. [65] studied the effect of postoperative probiotic supplementation on quality of life in CRC patients (individuals with sporadic CRC stages 0, I, II, III, who are undergoing confined colorectal resection surgery). e intervention of a combination of B. longum, L. acidophilus, and E. faecalis (each 1 × 10 7 CFU per gram) for 12 days (5 days before surgery and 7 days after surgery) improved the bowel function and reduced the incidence of diarrhea in CRC patients compared to the placebo group [65].
He et al. [66] conduced a meta-analysis of randomized controlled trials to investigate the effect of perioperative probiotic or synbiotic supplementation in CRC patients (individuals with CRC who are undergoing colorectal resection surgery). e perioperative administration of probiotic or synbiotic regimen significantly reduced the incidence of diarrhea, pneumonia, and total infection in CRC patients. Additionally, probiotic or synbiotic supplementation improved the microbiota by increasing the Lactobacillus and reducing the Enterobacteriaceae members, but no significant changes were observed in length of hospital stay, incision and perineal infection, septic morbidity, and anastomotic leak [66]. Some of the recent metaanalysis studies revealed that the supplementation of probiotic preparations consists of Lactobacillus strains effectively reducing the surgical inflammation and promoting the surgical recovery in CRC patients [67], and the probiotic supplementation also effectively reduced the postoperative infection and complications such as incision infection, pneumonia, and flatus time [68] and also improved the intestinal mucosal barrier function in CRC patients [69].
Kotzampassi et al. [70] studied the effect of postoperative probiotic supplementation on the postsurgery-related infectious complication and quality of life in CRC patients (individuals with CRC who are undergoing colorectal surgery). e perioperative supplementation of LactoLevure ® (B. lactis; 1.75 × 10 9 CFU, L. acidophilus; 1.75 × 10 9 CFU, Evidence-Based Complementary and Alternative Medicine L. plantarum; 0.5 × 10 9 CFU, and Saccharomyces boulardii; 1.5 × 10 9 CFU; twice per day) for 16 days (1 day before surgery, and 15 days after surgery) significantly reduced the postoperational complications compared to the placebo group. Specifically, the incidence of surgical site infection, anastomotic leakage, and pneumonia have been notably lower in the probiotic-supplemented group compared to placebo. Moreover, the expression of IL-6, TNF, and SOCS3 (suppressor of cytokine signaling 3) have been altered in a positive way to improve the quality of the postoperational life in CRC patients [70]. eodoropoulos et al. [71] investigated the effect of postoperative synbiotic supplementation on the postsurgery-related infectious complication and quality of life in CRC patients (individuals with CRC stages 0, I, II, III, and IV, who are undergoing colorectal surgery). e supplementation of synbiotic preparation (Synbiotic Forte ™ , probiotics includes Pediococcus pentosaceus, Leuconostoc mesenteroides, L. paracasei, L. plantarum, and prebiotics such as inulin, pectin, β-glucan, and resistant starch) for 15 days considerably improved the Gastro-intestinal Quality of Life Index and functional bowel disorder score in CRC patients compared to the placebo group, while no changes were observed in "constipation" score. e study revealed that the supplementation of Synbiotic Forte ™ improved the health condition of CRC patients after surgery, especially enhanced the gastrointestinal function [71] (Table 3).

A Possible Mechanism Underlying Anti-CRC Activity of Probiotics and Its Derivatives.
Even though several studies attempted to explain the mechanism of the anticarcinogenic property of probiotics [72][73][74][75], a clear mechanism behind the anti-CRC activity of probiotic has not been described yet. Several evidences revealed that probiotics confer the health benefits by modifying the composition of microbiota and its metabolic activities, production of anticarcinogenic and antimicrobial compounds, improvement of antioxidant system of the host, degradation of carcinogens, alter the expression of inflammation-associated genes, immune enhancement, and prevention of cancerous proliferation and apoptotic induction (Figure 1). Eventually, the continuous supplementation of any microbial preparation has an influence on the microbiota of the host. It has been proved that probiotic supplementation can positively alter the intestinal microbiota of the host system and aids to maintain the eubiosis [41,43]. e probiotics can produce antimicrobial substances (like bacteriocins), which hinder the growth of pathogenic microbes in the intestinal lumen, thereby preventing the dysbiosis and development of CRC [76].
Some of the bacterial enzymes (produced by the members of Clostridium, Bacteroides, Eubacterium) such as nitrate reductase, azoreductase, β-glucosidase, β-glucuronidase, and 7-α-dehydroxylase are associated with the production of carcinogenic compounds such as cresols, ammonia, phenols, aglycones, and N-nitroso compounds, and these compounds induce the antiapoptotic pathways, thereby facilitating the development of CRC [77,78]. Studies proved that the supplementation of probiotics reduced the activities of bacterial enzymes significantly [37,39,79,80]. e carcinogenic compounds bind with peptidoglycan, present in the cell wall, of the probiotic microbes and excreted through feces. Some of the probiotic strain can metabolize the carcinogenic compounds especially amines and N-nitroso compounds [76], and the alternation of metabolic activity (i.e., reduced the endogenous production of carcinogenic compounds) of intestinal microbiota and binding and degradation of carcinogens are some of the mechanisms by which probiotic supplementation reduced the risk of development of CRC. e compounds like short-chain fatty acids (SCFAs) such as butyrate, propionate, acetate, and conjugated linoleic acid (CLA) act as anticarcinogenic agents. Butyrate is a well-known SCFA associated with CRC. Lactic acid bacteria (LABs) do not produce butyrate but can convert the lactate and acetate into butyrate [76]. Most of the probiotic microbes are LABs. e supplementation probiotic will increase the concentration of SCFAs in the intestinal lumen that stimulated the release of antiinflammatory cytokines, suppressed the inflammatory pathways, and improved the antioxidant system [57,81,82]. Likely, CLA can induce the expression of PPAR-c, which influence the immune system, lipid metabolism, and apoptosis process [72,83]. e chronic inflammation is one of the lethal factors associated with the development of CRC, which disturb the intestinal microbiota [77,84]. e healthy intestinal microbiota is crucial for the maturation of the immune system and development of immunity against invading pathogens. e supplementation of probiotics aids to improve the immune system and modulate the immune system via regulating the secretion of anti-inflammatory cytokines and associated regulatory genes [78,85,86]. e improvement of intestinal permeability is often associated with several gastrointestinal tract associated illness. e probiotic supplementation improves the gut barrier function [87]. e intestinal epithelial line is protected by three important factors such as pH, tight junction proteins, and secreted mucins. e metabolic activity of probiotics produces several organic acids and SCFAs, which help to maintain the low pH in the intestinal lumen [88]. Probiotic supplementation improved the production and distribution of tight junction proteins such as occludin, claudin, and JAM-1 [87][88][89] and mucin production [90].
Antioxidant system is one of the major protective mechanisms of the host because free radicals are associated with several cellular damages and subsequent diseases. Several studies proved that the supplementation of probiotics improved the antioxidant status of the host [40,91]. e supplementation of probiotics alters the host physiology such as regulation of polyamines, ornithine decarboxylase enzyme activity [76,92], thereby reducing the risk of development of CRC.
Studies revealed that probiotic microbes can suppress the cancer cell proliferation and induce the apoptosis, which was mainly attributed to the production of SCFAs [93,94]. Evidence-Based Complementary and Alternative Medicine (1 × 10 7 CFU/g), L. acidophilus (1 × 10 7 CFU/g) and E. faecalis (1 × 10 7 CFU/g) 5 days before surgery and 7 days after surgery Improved the bowel function ↓ incidence of diarrhea [65] 12 Evidence-Based Complementary and Alternative Medicine e discussed mechanisms are affected by several factors such as probiotic strain, concentration, viability, duration of the consumption, and supplementation of dilatory fibers like prebiotics. us, not all the probiotics strains exhibit anti-CRC activities, it is necessary to screen the potent strain for the development of a probiotic-based therapeutic agent to control or prevent the incidence of CRC.
Evidence-Based Complementary and Alternative Medicine probiotics (L. rhamnosus GG or L. acidophilus [37]; L. plantarum [42]; L. casei BL23 [45] in vivo study) are some of the probiotic strains reported as adjuvant therapeutic agent to manage the CRC. Single-strain probiotic (L. rhamnosus GG along with guar gum fiber [52]) is reported as the promising adjuvant therapeutic agent to manage the CRC-related complications in CRC patients. Several studies evidenced that the probiotic (single genus and multispecies probiotics includes L. rhamnosus R0011 and L. acidophilus R0052 [62]; multigenus and multispecies probiotics includes L. casei, L. acidophilus, L. lactis, B. bifidum, B. longum, and B. infantis strains along with ω-3 fatty acid [53]; LactoLevure®, multigenus and multispecies probiotics includes B. lactis, L. acidophilus, L. plantarum, and S. boulardii [70]), synbiotic (Synbiotic Forte ™ , multigenus and multispecies probiotics includes P. pentosaceus, L. mesenteroides, L. paracasei, L. plantarum, and prebiotics such as inulin, pectin, β-glucan, and resistant starch [71]) intervention improved the health status of CRC patients after surgery. e beneficial impact of probiotic supplementation relay on the host physiology, disease severity, strain, dosage, duration of intervention, other food supplementations, etc. e probiotic supplements improved the immune system and intestinal integrity, increased the antimicrobial defense, and nullified the carcinogenic compounds in CRC patients. However, not all the probiotic interventions showed effective positive health effects in CRC patients. Further investigations are strongly recommended to reveal the exact mechanism and the potential of probiotics in CRC prevention.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
B. S. S conceptualized the study; B. S. S. and P. K. were responsible for methodology; B. S. S. wrote and prepared the original draft; B. S. S., P. K., and C. C. wrote, reviewed, and edited the manuscript; B. S. S., and C. C. supervised the work; B. S. S., P. K., and C. C. were responsible for project administration; and C. C. acquired funding.