الفهرس 
Review of LiteratureOnly 14 pages are availabe for public view
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Abstract
Cancer is considered one of the most lethal diseases affecting human health. It is regarded as the second leading cause of death throughout the world. Ehrlich ascites carcinoma (EAC) is a spontaneous mammary adenocarcinoma resembling human tumors. It has high sensitivity to chemotherapy; hence, it is widely used in cancer and chemotherapeutic studies.
Cisplatin (CDDP) is one of the most frequently used chemotherapeutic drugs with a broad spectrum efficacy against numerous human solid malignancies. As CDDP lacks tumor specificity, it can’t distinguish cancer cells from normal ones. Therefore it acts on DNA in the nucleus of normal and cancer cells forming DNA adducts with increased generation of reactive oxygen species (ROS). This leads to the interference with normal transcription and/or replication mechanisms triggering apoptosis and necrosis causing cell cycle arrest in normal and cancer cells. Cisplatin exhibits many adverse side effects including immunosuppression, myelo-suppression, hepatotoxicity, nephrotoxicity, ototoxicity, neurotoxicity, and cardio-toxicity as well as drug resistance.
Subsequently, the necessity to use adjuvant treatments to attenuate the chemotherapy toxicities and promote immune responses has become fundamental to overcome these obstacles. So recently, a close attention has been paid on health-promoting antioxidants from natural origins, which of economic and medical implications.
Milk has been a part and parcel in human nutrition. It has been used for decades by individuals of all ages due to its balanced composition of proteins, vitamins, lipids, and minerals.
Camel (Camelus dromedarius) milk (CM) has been used for centuries by nomadic people as complementary medicine especially in Arab countries due to its high biological value. It has been found to have several health benefits, including anti-tumor, antioxidant, anti-inflammatory, immune modulatory, hepato-protective hypoglycemic antiviral, antibacterial, ,and anti-fungal activities owing to presence of various bioactive components as lacto-ferrin (LF), lacto-peroxidase (LP), immunoglobulins (Igs) and natural antioxidants.
Goat (Capra hircus) milk (GM) is one of the neutraceutical health drinks. It is different from camel, cow, and human milk in compositions and nutritive value. It has several therapeutic benefits such as anti-inflammatory, antioxidant, anti-carcinogenic, immune-booster properties in addition to its easy digestibility with less allergic reactions due to presence of bioactive peptides, Zn, Mg, LP, LF, Igs antioxidants, high content of conjugated linoleic acid (anti-carcinogenic), and medium chain triglycerides.
The present study was conducted to investigate the effects of consuming CM and GM pre and post/ or post doses on alleviating the risks of cancer treatment by CDDP in cancerous female mice by measuring some of their bioactive components, some biological, biochemical, genetic and immunohistochemical evaluations, along with microscopic examinations of tumor and bone marrow (BM) tissues in all experimental mice groups.
Camel and goat milk had been analyzed for their major constituents of protein, fat, lactose, total solid non-fat and ash, in addition to determining some of their active bio-components; total immuno-globulins (Igs), lacto-peroxidase (LP) enzyme activity and total antioxidant capacity (TAC).
Data revealed that each 100 ml of CM contained; (3.1 ± 0.136g) protein, (1.99 ± 0.108 g) fat, (3.62 ± 0.221g) lactose, (6.44 ± 0.308g) total solid- nonfat and (0.61 ± 0.025 g ) ash. While each 100 ml GM contained; (3 ± 0.126 g) protein, (3.5 ± 0.282 g) fat, (3.68 ± 0.29 g) lactose, (5.99 ± 0.113 g) total solid nonfat, and (0.53 ± 0.037 g) ash.
In addition to the bioactive components of tested milk samples, total Igs (mg/dl) in CM and GM was (7810.1±4.322) and (3172.8 ± 4.486) respectively, LP enzyme activity (U/min/ml) was (0.447±0.042) and (0.346±0.014) in CM and GM respectively as well as TAC (mmol/L) in CM and GM was (5.76 ± 0.173) and (3.62 ± 0.119) respectively.
One hundred sixty adult female albino mice (BALB/c strain) weighing (22 ± 3 g) were offered standard commercial pellet diet and water ad libitum for 60 days after one week adaptation. All mice were randomly grouped into eight groups; each group contained 20 mice. Cancer was induced by intra-muscular (IM) Ehrlich ascites carcinoma (EAC) inoculation at dose of (2.5 × 106 viable EAC) in the left thigh of each mouse to induce Ehrlich solid carcinoma (ESC). Ten days after inoculating the EAC cells, the tumor became papale. CDDP was intra-peritoneally (I.P) injected at a single dose of (3.5mg/kg body weight). Fresh whole CM and GM were daily co-treated with CDDP by oral intra-gastric tube at dose of (0.4ml /20g mouse) as pre and post/ or post doses in tumor bearing mice groups. The eight groups were divided as the following; group I: Healthy control group; where mice were injected with physiological saline intra-peritoneally (I.P.) at a single dose. group II: Cisplatin group; mice were IP injected with CDDP at a single tested dose. group III: Cancer group; affected mice with intramuscularly (IM) injected EAC in the left thigh without treatment. group IV: Cancer + Cisplatin group; affected mice with IM injected EAC, were IP injected with a single tested dose of CDDP 10 days post EAC inoculation. group V: CM Neo-adjuvant-adjuvant group; (daily oral tested dose of CM for 2 weeks + IM inoculation of EAC + IP injection of CDDP after 10 days of EAC inoculation+ daily oral tested dose of CM for 2 weeks), group VI: GM Neo-adjuvant-adjuvant group; (daily oral tested dose of GM for 2 weeks + IM inoculation of EAC + IP injection of CDDP after 10 days of EAC inoculation+ daily oral tested dose of GM for 2 weeks), group VII: CM Adjuvant group; (IM inoculation of EAC + IP injection of CDDP after 10 days of EAC inoculation+ daily oral tested dose of CM for 2 weeks), and group VIII: GM Adjuvant group; (IM inoculation of EAC + IP injection of CDDP after 10 days of EAC inoculation+ daily oral tested dose of GM for 2 weeks). During the experimental period, feed intake was recorded and the animals were weighed weekly to monitor the body weight changes and to calculate the feed efficiency ratio (FER).
On the 25th day of Ehrlich solid carcinoma (ESC) inoculation; 10 mice from each group were weighed, sacrificed under anesthesia and blood samples were collected to perform the following biochemical evaluations; complete blood count (CBC), serum oxidative stress biomarkers (NO and MDA), antioxidant status (enzyme activities of serum CAT and RBCS SOD as well as blood GSH content), along with serum levels of inflammatory and anti- inflammatory cytokines namely; TNF α, IL-6 and IL-10 were also measured. As well as serum AST, ALT and γ GT enzyme activities were measured as liver function tests.
In addition to tumor and bone marrow (BM) were separated to perform the genetic study (P53, Bax, Casp-3, Bcl-2, Bax/Bcl-2 ratio, and comet assay in BM), immunohistochemical evaluations (PCNA gene expression in tumor), and physical biochemical tumor assessment. Moreover tissue histo-pathological examination of Ehrlich solid tumor and BM tissues from all experimental groups were accomplished to confirm the results of the previously mentioned evaluations.
The remaining mice (n = 10 mice) were left to complete the experimental period, to record their daily mortality rate, and to estimate MST and % ILS.
The results of present study are summarized as the following:
1- The biological data of the study reported that, there were a statistically significant (P≤ 0.05) reduction in body weight gain, feed intake, and FER in cisplatin and cancer groups while the maximum reduction was recorded in cancer + cisplatin group compared to healthy control group. However, oral consumption of CM or GM pre and post/ post doses caused a significant (P≤ 0.05) increment in body weight gain, feed intake, and FER with the most significant increment in mice received GM pre and post cancer treatment with CDDP by (+2.86 ± 0.643g), 90.56%, and (+ 0.0228 ± 0.00262) respectively compared to cancer + cisplatin group.
2- Regarding to hematological measurements; it was observed that there were statistically significant (P≤ 0.05) DROP in RBCs count, Hb content, Hct %, MCV, MCH, and MCHC in cisplatin and cancer groups with the greatest fall was found in cancer + cisplatin group by 32.07%, 46.65%, 39.04%, 10.27%, 21.47%,12.48% respectively compared to healthy control group. But the oral consumption of CM in CM neo adjuvant- adjuvant group revealed the highest significant (P≤ 0.05) improvement and increment in RBCs count (41.2%), Hb content (67.62%), Hct % (50.9%), MCV (6.87%), MCH (18.72%), and MCHC (11.07%) followed by oral consumption of GM in GM neo adjuvant- adjuvant group, after there, CM and GM adjuvant groups when compared to cancer + cisplatin group at (P≤ 0.05). Intra-peritoneal injection with CDDP in cisplatin group caused the highest significant fall (P≤0.05) in WBCs and PLTs counts as well as neutrophil % by 57.69%, 27.15% and 27.73% respectively followed by the IP injection with CDDP to cancerous mice in cancer+ cisplatin group by 32.75%, 18.48% and 13.53% respectively for the same parameters, while the highest significant DROP in lymphocyte % and monocyte % was observed in cancer + cisplatin group by 25.95% and 23.44% respectively followed by cancer group and the least significant fall was observed in cisplatin group as compared to their corresponding healthy control group. Whereas EAC induction in cancer group caused a significant rise (P≤0.05) in the total count of WBCs, PLTs and neutrophil % by 169.98%, 10.27%, and 11.99% respectively as compared to healthy control group. Camel milk consumption before and after EAC and CDDP induction caused the greatest significant improvement in total WBCs count, lymphocyte %, monocyte %, neutrophil %, and PLTs count by 39.52%, 32.2%, 29.47%, 11.76%, and 15.22% respectively, after that, GM consumption before and after EAC and CDDP induction then CM consumption after EAC and CDDP induction and the least significant improvement was reported in mice group consumed GM after EAC and CDDP induction as compared with their corresponding cancer + cisplatin group at (P≤0.05).
3- According the results of oxidative stress and antioxidant biomarkers; there was a statistically significant (P≤0.05) upsurge in serum NO and MDA levels while a remarkably declined serum CAT and RBCS SOD enzyme activities, along with blood GSH content in cisplatin, cancer, and cancer + cisplatin groups as compared to healthy control group. The highest elevation in serum NO and MDA levels was observed in cancerous mice treated with CDDP by 7.94 and 5.78 times respectively accompanied with greatest fall in serum CAT and RBCs SOD enzyme activities and blood GSH content by 53.99%, 71.5%, and 72.45% correspondingly in comparison with healthy control group at (P≤0.05). However, co-administration of CM and GM pre and/or post doses to cancerous mice treated with CDDP caused a significant (p≤0.05) improvement in oxidative stress and antioxidant biomarkers. CM neo-adjuvant-adjuvant group demonstrated the greatest significant (P≤0.05) improvement in serum levels of NO and MDA by 67.27% and 76.61% respectively associated with the highest enhancement in serum CAT and RBCS SOD enzyme activities, and blood GSH content by103.59%, 228.07%, and 219.11% respectively as compared to cancer+ cisplatin group.
4- Concerning serum inflammatory and anti-inflammatory biomarkers; there were a significant (P≤0.05) elevation in serum TNF-α and IL-6 levels associated with a significant DROP in serum level of IL-10 in cisplatin and cancer groups compared to healthy control group. Meanwhile the highest elevation in serum TNF-α and IL-6 levels were found to be 665.74% and 214.73% respectively accompanied with the greatest decrement in IL-10 level by 50.88% in cancerous mice treated CDDP compared to healthy control group at (P≤0.05). In contrast, mice co-treated with CM and GM pre and post/or post oral doses exhibited a significant (P≤0.05) improvement in serum TNF-α, IL-6 and IL-10 levels compared to GIV. CM neo adjuvant - adjuvant group showed the most significant (P≤0.05) amelioration in serum TNF-α and IL-6 levels by73.28% and 60.16% respectively and the most significant enhancement in serum IL-10 level by 88.41% compared to GIV.
5- With regard to serum liver enzyme activities, there were a significant (P≤0.05) rise in serum AST, ALT, and γGT enzyme activities in cisplatin and cancer groups with extreme upsurge in their activities by 232%, 245.15%, and 104.44% correspondingly in cancer +cisplatin group compared to healthy control group. Although, the co-administration of CM and GM as neo adjuvant and /or adjuvant oral doses showed a significant (P≤0.05) improvement in liver function enzymes compared with cancerous mice treated with CDDP. Mice co-treated with CM pre and post oral doses revealed the greatest hepato-protective effect with significant (P≤0.05) diminished serum enzyme activities of AST, ALT, and γGT by 60.8%, 54.128%, and 44.14% respectively compared to cancer + cisplatin group.
6- With respect to relative gene expression of apoptotic and anti-apoptotic biomarkers; the statistical analysis revealed a significant (P ≤ 0.05) up-regulation in the gene expression of P53, Bax, Casp-3, and Bax/Bcl-2 ratio associated with a significant down-regulation in Bcl-2 gene expression in BM of cisplatin, cancer, and cancer + cisplatin groups. Cancerous mice treated with CDDP showed the highest expression of P53, Bax, Casp-3, and Bax/Bcl-2 ratio by 6.93, 7.338, 8.028, and 50.659 folds respectively and the lowest gene expression of Bcl-2 by 85.51% as compared to healthy control group. However, the co-treatments of CM or GM had reversed the CDDP-induced BM apoptosis via depletion of P53, Bax, Casp-3 gene expression and Bax/Bcl-2 ratio, with subsequent enhancement in Bcl2 gene expression compared to cancer + cisplatin group. Pre and post CM co-treatment showed the most significant (P ≤ 0.05) improvement in BM relative gene expression of P53, Bax, Casp-3, and Bax/Bcl-2 ratio by 74.87%, 67.48%, 65.92%, and 94.68%, respectively with highest up-regulation of Bcl-2 gene in BM by 6.11 folds compared to cancerous mice treated with CDDP.
7- from the data obtained it was obvious that, CDDP injection in GII resulted in the greatest significant (P ≤ 0.05) DNA damage in BM cells which manifested by highly extended DNA tail moment, DNA tail length and DNA percent in tail by 1372.94%, 308.9%, and 260.23 % respectively, followed by cancerous mice treated with CDDP and cancerous mice groups compared to healthy control group. On the other hand, DNA damage of BM cells was significantly (P≤0.05) reduced through the co-treatment of CM or GM pre and post/or post oral doses with the best improvement by 27.69%, 12.38% and 17.47% for DNA tail moment, DNA tail length and DNA percent in tail, respectively in mice group received CM before and after cancer treatment with CDDP in comparison with cancerous mice treated with CDDP.
8- Regarding to tumor cell proliferation; the expression of PCNA using immunohistochemical staining was greatly increased in tumor tissues of cancer group by 2084.28% compared to healthy control group. However the lowest improvement in tumor tissue PCNA expression was reported in cancer + cisplatin group as it reduced by 21.15 %, while co-treatment of CM and GM pre and post/or post doses had enhanced CDDP anti-tumor efficacy with lesser side effects. The greatest improvement (lowest expression) was reported in CM pre and post co-treatment by 65.79% as compared to cancer group.
9- With regard to physical biochemical tumor study; there were a statistically significant reduction in tumor weight and tumor volume in different cancerous treated mice groups with the most significant reduction was found in CM neo adjuvant- adjuvant group by 75.25% and 84.35% respectively compared to cancer group at (P≤ 0.05). This led to a significant inhibition of tumor growth rate (TIR %) in GIV, GV, GVI, GVII, and GVIII by 28.51%, 66.89%, 60.46%, 53.79%, and 45.06% correspondingly in comparison with GIII. Moreover, both CM and GM pre and post/or post oral doses had caused a remarkable prolongation of MST with significant (P≤ 0.05) increased ILS% compared to cancer group. CM neo adjuvant-adjuvant group evidenced the highest prolongation of MST; (42 days) with ILS % of 68%.
10- The histo-pathological examination of solid tumor and BM tissues with Hematoxylin and Eosin (H & E) stains reconfirmed all the above findings. Microscopic examination of tumor tissues indicated that highest necrosis in solid tumor tissues with regenerated myofibers and reappearance of normal muscle structure was found in CM neo adjuvant – adjuvant group followed by GM neo adjuvant – adjuvant group, then CM adjuvant group, and after that GM adjuvant group, while the lowest the lowest significant necrosis was present in cancerous mice treated with CDDP only. Reflecting the role of CM or GM in alleviating CDDP side effects when treating cancer as well as their therapeutic role in killing tumor cells with enhancement of chemotherapeutic efficacy which may reduce drug dosage. Meanwhile, the mice injected with CDDP in either cisplatin group or in other cancerous mice groups severely affected BM tissue structure and caused a severe abnormal hematopoiesis. But the intra-gastric co-administration of CM and GM pre and post/ or oral doses had alleviated the side effects of CDDP treatment in cancerous mice groups with restoration of BM formation and noticeable hematopoiesis.
In general, the results of the current study showed that CM and GM could be used as adjuvant treatments with CDDP in treating cancer with minimal side effects due to their content of bio-active compounds and high antioxidant capacity.
Conclusion
from the results of the present study, it can be concluded that, camel and goat milk (CM and GM) are safe nutritional and immuno-modulatory agents. Their oral consumption could be an adjuvant food with cisplatin (CDDP) in cancer treatment, which could attenuate and prevent CDDP-induced risk factors.
Antioxidant, immuno-modulatory, and the anti-inflammatory potentials of CM and GM due to their nutritional and active content are the major mechanisms.
Pre and/ or post CM or GM co-administration caused a significant (p≤0.05) improvement in hematological profile, antioxidant status, reversed the CDDP-induced oxidative stress, inflammation and elevated liver enzyme activities. CM or GM co-treatment with CDDP resulted in a significant decreases (p≤0.05) in bone marrow (BM) tissues apoptosis, DNA damage and tumor cell proliferation. Also both milk enhanced the anti-tumor effects of CDDP by a reduction of tumor weight and volume with prolongation of mice life span. Microscopic examination of BM tissues and tumor masses confirmed these results.
Camel milk pre and post co-administration with CDDP recorded the most significant improvements followed by GM milk pre and post co-administration with CDDP.
Recommendations
The following recommendations are prescribed according to the results of this study:
1- Camel and goat milk consumption are considered as complementary diet as natural functional food recommended for the patients undergoing cancer treatment with chemotherapy.
2- Regular daily consumption of CM or GM (155.16 ml) approximately 2/3 of cup for reference man 70 kg (Paget and Barnes, 1964) as cancer patients treated with CDDP would help to decrease the severity of cancer treatment with CDDP. Camel and goat milk could be used as a potential functional food to prevent/protect against liver injury as a result of cancer treatment with CDDP.