Summary Alimentary tract (AT) mucositis can be a major problem for patients undergoing cancer treatment. It has significant clinical and economic consequences and is a major factor that can ...compromise the provision of optimal treatment for patients. The pathobiology of AT mucositis is complex and the exact mechanisms that underlie its development still need to be fully elucidated. Current opinion considers that there is a prominent interplay between all of the compartments of the mucosa involving, at a molecular level, the activation of transcription factors, particularly nuclear factor-κB, and the subsequent upregulation of pro-inflammatory cytokines and inflammatory mediators. The purpose of this review is to examine the literature relating to what is currently known about the pathobiology of AT mucositis, particularly with respect to the involvement of pro-inflammatory cytokines, as well as currently used animal models and the role of specific cytotoxic chemotherapy agents in the development of AT mucositis.
Purpose
Radiotherapy-induced gut toxicity (RIGT) is associated with diarrhoea, pain and rectal bleeding and can occur as an acute or chronic toxicity. The microvasculature has been shown to be ...altered in the development of RIGT; however, the features are not yet characterized. We hypothesized that apoptosis of microvascular cells would occur early in the gastrointestinal tract following fractionated irradiation, followed by late microvascular changes, including sclerosis and telangiectasis.
Methods
Female Dark Agouti rats were treated with a 6-week fractionated radiation schedule of 3 × 2.5 Gy doses per week localized to the abdomen. At 3, 6 and 15 weeks, the intestines were assessed for markers of acute and chronic injury including morphological changes, collagen deposition, apoptosis and proliferation.
Results
Apoptosis of microvascular cells significantly increased at 6 and 15 weeks in the jejunum (
p
= 0.0026 and
p
= 0.0062, respectively) and at 6 and 15 weeks in the colon (
p
< 0.0001 and
p
= 0.0005, respectively) in rats receiving fractionated radiation to the abdomen. Histopathological changes of the colon microvasculature were also seen from week 3, including thickening of the lamina propria and dilated, thickened, telangiectatic vessels.
Conclusions
Findings of this study provide evidence of regional and timing-specific changes in the intestinal microvasculature in response to fractionated radiotherapy which may play a role in development of both acute and chronic RIGT.
Purpose
Radiotherapy-induced gut toxicity (RIGT) is associated with significant diarrhoea, pain and rectal bleeding. Matrix metalloproteinases (MMPs) have been reported to be involved in ...chemotherapy-induced gut toxicity and RIGT following single-dose irradiation in vivo. We therefore proposed MMPs would be involved in the pathobiology of RIGT following fractionated irradiation.
Methods
Dark Agouti rats were treated with fractionated radiation (3 × 2.5 Gy/week for 6 weeks). Rats were killed at 3, 6 and 15 weeks to represent acute and chronic toxicities. Sections of jejunum and colon were immunostained for MMP-1, MMP-2, MMP-9 and MMP-14. Relative mRNA expression in jejunum and colon was quantified by RT-PCR for MMP-1, MMP-2, MMP-9 and MMP-14. Western blotting was also conducted on jejunum and colon tissue collected at week 6 to determine protein levels of pro- and active MMP-2.
Results
MMP-2 total protein levels, determined by western blotting, significantly increased in both the jejunum (
p
= 0.0359) and the colon (
p
= 0.0134) 6 weeks into the fractionated radiation schedule. MMP-1, MMP-2, and MMP-14 mRNA expression significantly increased in the jejunum. MMP-2 mRNA expression was also significantly increased in the colon. Immunostaining of MMP-2 was observed to be increased in both crypt enterocytes and the lamina propria.
Conclusions
MMP-2 plays a role in the pathobiology of gastrointestinal toxicities following fractionated irradiation. Whilst MMP-1 and MMP-14 mRNA expression was increased, this occurred only in the jejunum, suggesting MMPs are differentially involved in RIGT depending on the intestinal region. Further studies are needed to elucidate the role these mediators play in the development and potentiation of RIGT.
5-Fluorouracil (5-FU) is a commonly used chemotherapy agent in clinical oncology practice. Two of its major side effects are mucositis and diarrhoea. The structure of mucins offers mucosal ...protection, and allows maintenance of intestinal flora by providing attachment sites and preventing bacterial overgrowth and/or penetration. The aim of this study was to investigate changes in mucin secretion and microflora following treatment with 5-FU. Female DA rats were given a single 150 mg/ kg i.p. dose of 5-FU. Rats were killed at various time points after treatment. Control rats received no treatment. Jejunum, colon and faecal samples were collected. Standard microbiological culture techniques were used to identify bacteria, and real-time PCR was used to quantify bacteria in faecal samples. Goblet cells and cavitated goblet cells (having undergone mucus exocytosis) were also counted. Statistical analysis was carried out using Kruskal-Wallis test, a non-parametric method of testing equality of group medians. Following treatment with 5-FU, we showed decreases in Clostridium spp., Lactobacillus spp. and Streptococcus spp., and an increase in Escherichia spp. in the jejunum. In the colon, 5-FU caused decreases in Enterococcus spp., Lactobacillus spp. and Streptococcus spp. Real-time PCR of faecal samples showed decreasing trends in Lactobacillus spp. and Bacteroides spp., and an increasing trend in E. coli. Significant increases (P < 0.05) were seen in Clostridium spp. and Staphylococcus spp. at 24 h. Goblet cell numbers decreased significantly in the jejunum from 24-72 h, with a significant increase in the percentage of cavitated goblet cells. In conclusion, 5-FU treatment causes significant changes in intestinal flora and mucin secretion in rats. These changes could result in systemic effects and, in particular, may contribute to the development of chemotherapy-induced mucositis.
Background: One of the most common toxicities of cancer treatment is diarrhoea. Probiotics have been shown effective at preventing diarrhoea in inflammatory bowel disease and may prove useful in the ...oncology setting. Aim: The primary aim of this study was to investigate the probiotic mixture, VSL#3, for amelioration of chemotherapy-induced diarrhoea (CID). Methods: This experiment was carried out in a clinically relevant model of CID. VSL#3 was administered to female DA rats in one of three schedules. Irinotecan was used to induce mucositis and diarrhoea, with rats monitored for 7 days to record incidence of weight-loss and diarrhoea. At study completion, intestines were collected to investigate histological and proliferative changes, apoptosis levels and mucin composition. Results: VSL#3 reduced weight loss following irinotecan when administered before and after chemotherapy. Moderate and severe diarrhoea was also prevented in these rats. This was associated with a significant increase in crypt proliferation combined with an inhibition of apoptosis in both the small and large intestines. VSL#3 also prevented irinotecan-induced increases in goblet cells within jejunal crypts. Conclusions: VSL#3 is effective at preventing severe diarrhoea following chemotherapy with irinotecan and therefore has potential to be used clinically by cancer patients.
Mucositis is a toxic side effect of anti-cancer treatments and is a major focus in cancer research. Pro-inflammatory cytokines have previously been implicated in the pathophysiology of ...chemotherapy-induced gastrointestinal mucositis. However, whether they play a key role in the development of radiotherapy-induced gastrointestinal mucositis is still unknown. Therefore, the aim of the present study was to characterise the expression of pro-inflammatory cytokines in the gastrointestinal tract using a rat model of fractionated radiotherapy-induced toxicity.
Thirty six female Dark Agouti rats were randomly assigned into groups and received 2.5 Gys abdominal radiotherapy three times a week over six weeks. Real time PCR was conducted to determine the relative change in mRNA expression of pro-inflammatory cytokines IL-1beta, IL-6 and TNF in the jejunum and colon. Protein expression of IL-1beta, IL-6 and TNF in the intestinal epithelium was investigated using qualitative immunohistochemistry.
Radiotherapy-induced sub-acute damage was associated with significantly upregulated IL-1beta, IL-6 and TNF mRNA levels in the jejunum and colon. The majority of pro-inflammatory cytokine protein expression in the jejunum and colon exhibited minimal change following fractionated radiotherapy.
Pro-inflammatory cytokines play a key role in radiotherapy-induced gastrointestinal mucositis in the sub-acute onset setting.
Purpose Mucositis is a major oncological problem, caused by the cytotoxic effects of cancer chemotherapy and radiotherapy. Irinotecan is used to treat a variety of solid tumours, through the ...inhibition of DNA topoisomerase I and is linked with severe mucositis and diarrhoea. Mucus production appears to be increased, which may contribute to the development of diarrhoea. Methods Dark agouti rats were treated with irinotecan, and tissues collected at several time points up to 72 h. Goblet cells and mucin secretion were investigated, as well as mucin expression (Muc2 and Muc4) and kruppel-like factor (Klf) 4 using immunohistochemistry in the gastrointestinal tract. Both goblet cells and cells positive for Muc expression were counted, and analysed statistically using the Mann-Whitney U test with Bonferroni correction. Results Goblet cells decreased significantly after irinotecan treatment. However, mucin secretion increased. Mucin expression changed significantly after treatment. Muc2 and Muc4 decreased significantly in the villi of the jejunum after treatment, Muc2 and Muc4 decreased significantly in the crypts. Muc2 decreased significantly in the colon. Conclusions Irinotecan causes an increase in mucin secretion and a net decrease in mucin-producing goblet cells, and the expression of Muc2 and Muc4 in the gastrointestinal tract is altered following treatment. Increased mucin secretion is likely to be related to altered mucin expression, and may contribute to chemotherapy-induced diarrhoea.
Summary
Chemotherapy‐induced diarrhoea is a major oncological problem, caused by the cytotoxic effects of cancer chemotherapy. Irinotecan is linked with severe mucositis and diarrhoea, the mechanisms ...of which remain poorly understood. Bacterial β‐glucuronidase is thought to be involved in the metabolism of irinotecan, implicating the intestinal flora. Intestinal mucins may also be implicated in the development of chemotherapy‐induced diarrhoea. Rats were treated with 200 mg/kg of irinotecan and killed at 96, 120 and 144 h. The rats were monitored for diarrhoea. Pathology and immunohistochemical staining was performed. The samples were cultured and faecal DNA was analysed using real‐time polymerase chain reaction. Severe diarrhoea was observed from 72 to 96 h. A decrease in body mass was also observed after treatment. Significant changes in goblet cell numbers (both complete and cavitated cells) were observed in the small and large intestines. Changes in MUC gene expression were observed in the small intestine only. Modifications were observed to the intestinal flora profile, especially Escherichia coli, and an increase in the expression of β‐glucuronidase was detected. In conclusion, irinotecan‐induced diarrhoea may be caused by an increase in some β‐glucuronidase‐producing bacteria, especially E. coli, exacerbating the toxicity of active metabolites. Accelerated mucous secretion and mucin release may also contribute to the delayed onset of diarrhoea.
Radiation-induced mucositis is a common and serious side effect of radiotherapy. Molecular mechanisms of mucosal injury, however,
are still poorly understood and extremely difficult to study in ...humans. A novel Dark Agouti rat model using fractionated radiotherapy
to induce mucositis has been developed to investigate the occurrence of alimentary mucosal injury. Twenty-four Dark Agouti
rats were randomly assigned to receive either fractionated radiotherapy or no radiotherapy. The irradiated rats received a
fractionated course of abdominal radiotherapy at 45 Gy/18 fractions/6 weeks treating thrice weekly (i.e., at a radiation dose
of 2.5 Gy per fraction). After each week of radiation, a group of irradiated rats was killed. Histomorphology and mucin distribution
in the alimentary tract was investigated. The terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay
was used to examine apoptosis in the colon and jejunum, and intestinal morphometry was used to assess villus length, crypt
length, and mitotic crypt count. Immunohistochemistry of p53, nuclear factor-κB, cyclooxygenase (COX)-1, and COX-2 was also
done. The fractionated radiotherapy course induced alimentary mucositis from week 1, with more severe injury seen in the small
intestine. The hallmark appearance of apoptosis was present in the crypts of the small and large intestine. In the jejunum
and colon, goblet cell disorganization and degeneration was obvious and crypt mitotic counts were severely depleted throughout
the treatment. Expression of p53, nuclear factor-κB, COX-1, and COX-2 was increased in the irradiated intestinal sections.
Fractionated radiation-induced alimentary mucositis has been effectively documented in the Dark Agouti rat for the first time.
Further studies investigating the molecular mechanisms underlying radiation-induced mucositis are planned to ultimately achieve
anti–mucotoxic-targeted therapies. Mol Cancer Ther 2007;6(8):2319–27
Mucositis is a common, costly and unpleasant side effect of cancer chemotherapy and radiotherapy. Velafermin (FGF-20) has shown the potential to reduce these side effects. Irinotecan is a ...chemotherapeutic agent which is commonly used in solid Tumors, and causes GI mucositis manifested by severe diarrhoea. Therefore the primary aim of this study was to investigate whether velafermin reduces the GI mucositis induced by irinotecan. The secondary aim was to test varying schedules of administration of velafermin. Groups of Tumor-bearing DA rats (6 per group) were treated with varying doses (4, 8 or 16 mg/kg) of velafermin intraperitoneally either prior to, prior to and during, or after chemotherapy treatment. Rats received a single dose of 200 mg/kg irinotecan intraperitoneally. Rats were monitored closely for the incidence and severity of diarrhoea and mortality before being killed 192 h following treatment. Mortality, diarrhoea and histopathology were assessed throughout the gastrointestinal tract. Severe or moderate diarrhoea occurred in approximately 40% of rats treated with irinotecan alone. This was associated with a 50% mortality rate 96 h following chemotherapy. Velafermin administered at 16 mg/kg prior to irinotecan improved gastrointestinal mucositis as measured by reduced diarrhoea and mortality following irinotecan chemotherapy in the DA rat. Rats that received velafermin prior to, or prior to and during irinotecan treatment did develop severe or moderate diarrhoea, however it occurred later, in fewer rats and was not associated with mortality. Other dosing regimens were not as effective. This has important implications for the use of velafermin in GI mucositis in humans, and should be further studied.
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