Colchicine Enhances the Apoptotic, Anti-tumor Efficacy, Survival of Doxorubicin and Lowers Associated Toxicity in an Ehrlich Ascites Cancer Mouse Model

Doxorubicin (DOX) is an effective chemotherapeutic drug but induces serious adverse effects. The anti-inflammatory drug colchicine (COL) was found to inhibit inflammasome activity involved in DOX side effects. The purpose of this research is to investigate COL impact when being added to DOX if decreases side effects or enhances its anti-tumor efficacy. To this end, we used Ehrlich ascitic carcinoma (EAC)-bearing CD1 female mice and treated them with high and low doses of DOX (DOX high and DOX low ) in the presence or absence of COL. Mice were inoculated intraperitoneally with 0.25 × 10 6 EAC-cells/mouse and then treated with DOX high (2 mg/kg), DOX low (1 mg/kg), COL (5 µmol/kg), DOX high /COL and DOX low /COL. On day 8 of tumor injection, 50% of the mice were sacrificed to evaluate tumor volume, total tumor cell count, EAC cell apoptosis, cell cycle, hematological, and biochemical parameters, including liver and kidney function tests, oxidative stress (OS) markers, C-reactive protein (CRP), and interleukin 1-beta (IL-1β). The remaining 50% of mice were left to determine the survival of the groups. Co-treatment of COL with DOX high or DOX low enhanced the overall antitumor effect of DOX as evidenced by an enhancement in the tumor parameters, an increase in EAC cell apoptosis, and induction of cell cycle arrest. Additionally, their co-treatment ameliorated DOX adverse effects as evidenced by an improvement in the measured markers. Conclusion : Combination of COL with DOX high or DOX low enhanced the antitumor effect and decreased the adverse effects. This study opens a new avenue to their use in the clinical setting.


Introduction
Doxorubicin (DOX) (PubChem CID: 31703) https://pubchem.ncbi.nlm.nih.gov/compound/31703 is an anthracycline antibiotic extracted from the bacterium Streptomyces peucetius [1].It is usually prescribed for the management of various types of tumors [2].DOX performs its anticancer activity by several pathways including DNA intercalation and topoisomerase II inhibition, histone expelling, production of reactive oxygen species (ROS), overproduction of ceramide, and modulation of calcium and iron homeostasis [3].Although effective as an anti-cancer therapy, DOX has many side effects as bone marrow suppression, alopecia, bladder problems, and liver and kidney toxicities [4].Being an essential organ for the metabolism and detoxification of several drugs, both the liver and kidney are the primary body organs that are mostly affected by chemotherapy in cancer patients [5].Hepatic cells are among the various tissues of the body in which DOX is accumulated [6].Moreover, DOX increases proteinuria, plasma creatinine, and glomerular capillary permeability in the kidney [7].Furthermore, DOX alters the cell apoptotic signaling via increasing the pro-apoptotic protein Bax expression and decreasing the anti-apoptotic protein Bcl2 expression in cancer as well as in healthy cells [7].Finally, and most importantly, tumor relapse may occur due to resistance to DOX [8].One of the main mediators of DOX resistance and DOX role during cardiotoxicity is the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome pathways that function both in cancer and cardiac cells [9].The NLRP3 inflammasome is a cytoplasmatic multiprotein complex belonging to the innate immune system, consists of receptors, adaptor protein (apoptosis-associated specks-like protein containing a CARD (ASC)), and caspase-1 that triggers pro-IL-1β and pro-IL-18 conversion into their activated cleaved forms [9, 10].Beyond IL-1β and IL-18 production, NLRP3 stimulation results in the formation of the gasdermin D (GSDMD) pore and finally pyroptosis [10].In general, NLRP3 inflammasome has a pivotal role in a broad spectrum of diseases, including cardiac diseases [11] and cancer [9].In cancer, NLRP3 and IL-1β drive cancer progression involving tumorigenesis promotion, angiogenesis, immunosuppression, and metastasis [12,13].Colchicine (COL) (PubChem CID: 6167) https://pubchem.ncbi.nlm.nih.gov/compound/6167 originally isolated from Colchicum autumnale, is a tricyclic alkaloid drug [14] clinically used for inflammatory diseases treatment as the gouty arthritis and familial Mediterranean fever (FMF) [15].COL binds to an unpolymerized tubulin [16], resulting in the inhibition of microtubule polymerization [17].This explains the potent antitumor activities of COL and its derivatives [18].COL antitumor activity could, also, be mediated by its modulatory effect on immune cells, leading to the development of effective cancer immunotherapies [19].Moreover, COL inhibits tumor necrosis factor-alpha activity, leukotriene B4, prostaglandin E2, thromboxane A2, and cyclooxygenase-2 [15].Interestingly, COL also inhibits NLRP3 inflammasome [15,20] by inhibiting caspase-1 and transport of ASC as well as the expression of the pyrin gene responsible for NLRP3 receptor expression, IL-1β, and P2X7, resulting in an increased intracellular potassium [15].Interestingly, COL treatment has been shown to ameliorate cardiotoxicity induced by chemotherapeutic drugs such as 5-fluorouracil in rats [21].Therefore, COL might ameliorate toxicities in other body organs.Given these unique biological effects of COL, it could be used as a potential anti-cancer drug with potential antitoxic effects.
Study aim: to investigate whether or not COL can enhance the antitumor activity of DOX and lower DOX used dose as well as its associated toxicities.

Animals
The research committee of ethics (REC) agreed on the protocol (ENREC-ASU-2020-7).The research was conducted with minimal harm to animals following the ARRIVE guidelines for animal handling and welfare.CD-1 ® IGS Mice: female white Swiss albino mice bred using the Charles River (Crl) International Genetic Standardization (IGS) system.Crl: CD1(ICR): Charles River CD1 Institute for Cancer Research (ICR). 2 months, 25±3 g BW female mice purchased from the National Institute of Cancer (NCI, Cairo, Egypt) initially imported from the USA.Mice were housed in the facility of animals at Pharmacy Faculty, University of Ain Shams for a week for adaptation, with accommodation, and relative humidity was kept at 22±1.0 C and 55±5.0%,correspondingly, as well as light-dark cycles.Tap water and standard pelleted animal food were provided to mice ad libitum.

Experimental design was done on the top of a Pilot Study
A pilot experiment was conducted to study the additive and the mechanistic impact(s) of giving COL with DOX in an experimental tumor mouse model (EAC), to decide whether it is better to administer DOX and COL simultaneously or not.Blood was collected from mice to measure CRP and CBC.

Experimental Design
After 1 week of preparation, in the animal house at the Faculty of Pharmacy, Ain Shams University, 140 mice were, randomly divided, using a random number generator https://www.graphpad.com/quickcalcs/randomize1/, into 7 groups (N = 20/group) [23, 24], (to minimize the standard errors between groups) as shown: Group 1: mice were treated with 200 µL of sterile PBS i.p. injected on days 3, 5, and 7 (control), From Group 2 to Group 7, mice were inoculated with 0.25 × 10 6 of EAC-cells/mouse i.p. on Day 0 as follows: [N.B. for comparison, tumor growth in PBS-treated control animals was taken to be 100%].

EAC-cells inoculation
2 stock mice injected with EACs, provided by the National Institute of Cancer (NCI, Cairo University, Egypt), after 7 days post-EAC's challenge were used as a source for EAC cells.EAC cells were obtained from the mice and the count was adjusted to 0.25×10 6  On the eighth day of EAC tumor inoculation, first, 50% of all groups were anesthetized to obtain samples of blood for hematological and biochemical analysis.Blood was obtained from mice for sera separation as previously described [26], then sera were kept aliquoted at -80 C.Second, the sacrifice of mice was performed by cervical dislocation to harvest the ascetic tumor fluid to determine the total ascite volume and the total live and dead tumor cell count.Early, late apoptotic, and necrotic percentages (%) of EACcells and EAC-cell cycle analysis were determined via flow cytometry (FC).Third, the liver was extracted and homogenized in PBS to measure the oxidative stress markers.The remaining 50% of mice in each group were left alive, from day 8 to day 24 for mice survival rate determination (endpoint).No humane endpoints were established.The study included all the animals with no exclusions.

Determination of the % of body weight changes
The percentage of body weight changes (% BW) was calculated as previously described [27].

Determination of the total tumor volume and count
Tumor volume and count were determined as previously described [27].

Estimation of Animal Survival
The effect of different treatment protocols on survival was monitored until day 24 (endpoint) after EAC cell inoculation (day zero), by recording mortality daily of 50% of mice (N= 10/group) for the Kaplan Meir curve.

Apoptosis Analysis of EAC-cells
Annexin V-FITC was used to analyze the apoptotic cells (Becton Dickinson BD Pharmingen TM , Heidelberg, Germany) in the ascetic fluid cell harvest.Shortly, EAC-cells were obtained from EACharboring mice in the different groups and washed two times in PBS for 20 min each.Next, EAC-cells from untreated and treated groups were re-suspended in 100 µL of binding buffer with the introduction of 1 µL of FITC-Annexin V, then 40 min.keeping at 4 °C.Cells were then rinsed and re-suspended in 150 µL of binding buffer with the introduction of 1 µL of PI.Then, the FC BD FACS Caliber (BD Biosciences, San Jose, CA, USA) was employed for analysis.

Analysis of EAC-cells cycle
Additionally, CycleTEST TM PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA) was used to perform the analysis.The EAC-cells from untreated and treated groups were stained with PI (Becton Dickinson BD Pharmingen TM , Heidelberg, Germany) as described by the kit and then run on the cytometer.CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) was employed to study the cell cycle distribution [28].

Determination of Blood Parameters
Hematological parameters were analyzed as described earlier [29].

Determination of Biochemical Parameters
Serum ALT, AST, lipid profile, urea, creatinine, and CRP in addition to mouse IL-1β levels determination were performed using appropriate kits.The liver was extracted and homogenized in PBS to measure the SOD and CAT enzyme activities and MDA levels.

Statistical Analysis
Scatter plots with bars were employed to represent the results showing the respective mean ± S.D. One-way ANOVA test and Tukey's post hoc test were employed.All analyses, including the Kaplan-Meier survival curve, were performed using GraphPad Prism version 8.00 (GraphPad Software, San Diego California USA).At a pvalue less than 0.05, results are considered significantly different.

Colchicine lowers the doxorubicininduced decrease in body weight
Initial body weight (initial BW), final body weight (final BW), and the percent change in body weight are shown in Table 1.After 8 days of EAC-cells inoculation in mice, the final BW. was 1.2-fold (P<0.0001)greater in EAC mice when put in comparison with negative control mice.Administration with DOX high or DOX high /COL led to a 1.5-fold (P<0.0001) and 1.4-fold (P<0.0001)decrease in the final BW, correspondingly, relative to the positive control.
Treatment of EAC-mice with COL reduced the final BW by 1.1-fold (P<0.01) when put in comparison with positive control.Treatment of EAC-injected mice with DOX low or DOX low /COL decreased the final BW by 1.1-fold (P<0.001) and 1.2-fold (P<0.0001)correspondingly, when put in comparison with the positive control.Taken together, the combination of COL with DOX lowered the decrease in BW.

Colchicine increases the doxorubicininduced antitumor effects
Tumor parameters are presented in Table 2. EAC-harboring mice treated with DOX high and COL exhibited an 8.1-fold (P<0.0001) and 1.8fold (P<0.0001)decrease in the total tumor volume (the total ascitic fluid volume) when contrasted with EAC-bearing mice, respectively.
Administration with DOX high /COL showed the highest effect (18.3-fold decrease; P<0.0001).DOX low or DOX low /COL treated EAC-injected mice revealed a 2.6-fold (P<0.0001) and 4.3-fold (P < 0.0001) decrease in the total tumor volume relative to EAC-harboring mice, correspondingly.EAC-bearing mice treated with DOX high showed a 14.8-fold reduction (P<0.0001) in the total tumor count (25±1.6 x 10 6 /mouse) when compared to EAC-bearing mice (370 ± 6.98 x 10 6 /mouse).COL-treated EAC-bearing mice showed a 3.7-fold (P<0.0001)decrease in the total tumor count (100±3.4x 10 6 /mouse) when opposed to EAC-injected mice.Administering a combination of DOX high /COL showed the highest reduction in the total tumor count; leading to 37-fold (P<0.0001)lower tumor cells when put in comparison with the control group.The treatment of EAC-injected mice with DOX low or DOX low /COL led to a 6.2-fold (P<0.0001) and 10.6-fold (P<0.0001)decrease in the total tumor count in comparison with the control group.Compared to mice given DOX low , mice that were given a combination of DOX low and COL showed a 1.7-fold (P<0.0001)greater decline in the total tumor count (35±3.4x 10 6 vs 60±3.2 x 10 6 /mouse).Compared to the control group, all the treated groups showed a major reduction in the total live EAC-cells (P<0.0001).Taken together, COL optimizes the antitumor effects induced by DOX.Values represent the mean ± SD of tumor volume/mouse (mL), total tumor count (×10 6 /mouse), live cells count (×10

Doxorubicin and colchicine extend the survival of EAC-injected mice
To assess the impact of different treatments on the survival of EAC-harboring mice, we monitored the survival until day 24 after EAC cell inoculation.On day 12 after EAC inoculation, 100% of the untreated mice died.
COL-treated EAC-harboring mice showed extended survival until day 17, whereas 100% of EAC-injected mice given DOX high were still alive at day 24 post-EAC inoculation.In contrast, only 85.7% of EAC-injected mice given DOX high /COL or DOX low /COL were alive on day 24 after EAC injection, and only 57.1% of mice treated with DOX low (Fig. 1).

Cotreatment of doxorubicin with colchicine increases the number of early, late apoptotic, necrotic EAC-cells and EAC cell arrest at the G2M phase
The % of necrotic, early apoptotic, and late apoptotic EAC-cells obtained from EAC-injected mice were 0.5±0.1%,1.9±0.4%,and 0.8±0.2%,respectively.Early and late apoptotic EAC cell percentages harvested from mice treated with DOX high or COL were 7.5-fold (P<0.05) and 4.7fold (P<0.05)higher, correspondingly, than those of mice injected with EAC cells only.However, treatment with a combination of DOX high and COL did not show significant differences in the percentages of those cells when put in comparison with DOX high -treated mice.Coadministration of DOX low and COL increased the apoptotic cell percentage by 1.6-fold (P<0.05)contrasted with those harvested from DOX lowtreated mice (Fig. 2a).
EAC-cells Cell cycle Analysis from EACbearing mice showed that the % of G1, S, and G2/M phases were 55±1.7 %, 30±1.0 %, and 15± 2.0 %, respectively.% of cells harvested from DOX high -treated EAC-bearing mice was 1.3-fold (P<0.001)lower at the G1 phase, while 1.5-fold (P<0.0001)greater at the S phase, relative to mice injected with EAC cells only.The percentage of EAC cells in the G2/M phase did not differ between EAC cells from DOX hightreated mice and EAC cells from untreated EACinjected mice.The % of EAC cells from COLadministered mice was 1.3-fold (P<0.001)lower in G1, while 2.0-fold higher (P<0.001) in the G2/M phase, contrasted with control EAC cells.COL did not alter the % of EAC cells in the S phase.However, treatment with DOX high /COL reduced the % of cells in the G1 phase by 1.7fold (P<0.0001) and increased cells at the G2/M phase by 2.0-fold (P<0.0001) and at the S phase by 1.2-fold (P<0.01)relative to EAC-harboring mice group.Percentages of cells at G1 and G2M phases harvested from DOX low -treated EACbearing mice were not significantly different from those in control EAC cells.By contrast, the % of cells at the S phase was 1.3-fold (P<0.01)higher.Administration with DOX low /COL showed a 1.4-fold (P<0.0001)decrease in the % of EAC cells at the G1 phase and a 1.7-fold (P<0.01)increase in the % of EAC cells at G2/M compared to control EAC cells (Fig. 2b).% of cells at the G2M phase was 2.2-fold (P<0.0001)greater in the DOX high /COL group compared to the DOX high group, whereas it was 2.1-fold (P<0.001)greater in the DOX low /COL group relative to DOX low group.

Colchicine alleviates tumor-associated hematological toxicity
Total RBC count, Hb concentration, and Hct % were 1.5-fold (P<0.05)lower in EACpossessing mice contrasted with the data of the control mice (Table 3).Administration of DOX high to EAC-possessing mice increased these factors by 1.6-fold (P<0.05),1.4-fold (P<0.05), and 1.4-fold (P<0.05),sequentially, contrasted with untreated EAC-possessing mice.COL treatment of EAC-bearing mice increased those parameters by 1.7-fold (P<0.05),1.3-fold (P<0.05), and 1.3-fold (P<0.05),respectively.The combination of DOX high with COL raised total RBC count, Hb concentration, and Hct % by 1.5-fold (P<0.05),1.4-fold (P<0.05), and 1.6-fold (P<0.05)relative to EAC-bearing mice.The number of platelets was slightly decreased in EAC-possessing mice relative to the control group.This count was also slightly decreased after the treatment with DOX high or after combination with COL or COL alone relative to EAC-harboring mice.Total WBCs count increased by 2.8-fold (P<0.0001) in EAC-bearing mice contrasted with control mice.EAC-bearing mice treated with DOX high , COL or DOX high /COL or DOX low or DOX low /COL displayed 1.5-fold (P<0.0001),1.2-fold (P<0.001),1.9-fold (P<0.0001),1.3-fold (P<0.0001), and 1.5-fold (P<0.0001)lower total WBCs count compared to control EAC-bearing mice, respectively.Collectively, COL induced alleviation in the hematological toxicities associated with the tumor as well as tumor treatment when combined with DOX.

Co-treatment of doxorubicin with colchicine ameliorates the lipids profile
The lipids profile of the different mice groups is presented in Table 4. Serum TC, TAG, and LDL-C values were 2.0-fold (P<0.0001),1.6-fold (P<0.0001), and 2.3-fold (P<0.0001)higher, correspondingly, while the HDL-C values were 1.8-fold lower (P<0.0001) in EACharboring mice relative to the control group.Treatment of EAC-harboring mice with DOX high or DOX high /COL or DOX low /COL resulted in a notable decrease in the TC and TAG values.Treatment with COL or DOX low , however, significantly decreased TC levels by 1.2-fold (P<0.0001) and 1.1-fold (P<0.001),correspondingly, but not the TAG levels, as opposed to the EAC-carrying mice group.
Subjecting EAC-carrying mice to DOX high or COL or DOX low or DOX low /COL did not alter LDL-C levels.Only treatment with DOX high /COL decreased LDL-C levels by 1.3-fold (P < 0.001) relative to the EAC-bearing group.None of the treatments induced changes in HDL-C levels.In conclusion, co-treatment of COL with the high dose of DOX improved the lipids profile.

Co-treatment of colchicine with doxorubicin decreases the tumor-associated oxidative stress
Tumor burden decreased the activities of SOD and CAT by 4.1-fold and 2.5-fold at P<0.0001, correspondingly, but increased those of MDA by 2.7-fold (P<0.0001).Treatment of EAC-bearing mice significantly increased SOD and CAT activities and decreased MDA levels in all treatment groups relative to the EACharboring mice group.Interestingly, administering DOX high /COL improved SOD and CAT activities by 1.3-fold (P<0.01) and 1.4-fold (P<0.0001),correspondingly, and decreased MDA levels by 1.5-fold (P<0.0001) when contrasted with EAC-carrying mice that received DOX high .In contrast, treatment of EAC-carrying mice with DOX low /COL only led to a 1.1-fold (P < 0.05) decline in MDA levels when contrasted with the DOX low-treated group.Therefore, cotreatment of DOX with COL decreases the oxidative stress (Fig. 4 a, b, and c).

Colchicine decreases doxorubicin-induced inflammation
CRP and IL-1β are known as typical inflammatory mediators.Our results indicate that serum CRP and IL-1β were 5.7-fold (P<0.0001) and 3.6-fold (P<0.0001)greater in EAC-bearing mice versus control mice, sequentially.DOX hightreated EAC-harboring group further showed a 1.4-fold (P<0.0001) and 1.2-fold (P<0.0001)elevation in CRP and IL-1β levels, sequentially, while COL-treated EAC-harboring group showed a 1.8-fold (P<0.0001) and 1.4-fold (P<0.0001)decline, correspondingly.Although co-treatment with DOX high and COL did not change CRP, they decreased the IL-1β levels by 1.2-fold (P<0.0001) when contrasted with EAC-harboring mice.While subjecting EAC-harboring mice with DOX low did not affect the levels of CRP and IL-1β, treatment with DOX low /COL resulted in 1.1fold (P<0.001)lower IL-1β levels.Administration of DOX high /COL resulted in a 1.2fold (P<0.01) and 1.5-fold (P<0.0001)decline of CRP and IL-1β, sequentially, when contrasted with EAC-harboring mice that received DOX high only.Whereas their treatment with DOX low /COL decreased IL-1β levels by 1.2-fold (P<0.0001) when contrasted with EAC-bearing mice that received DOX low .In conclusion, COL decreases DOX-induced inflammation (Fig. 4 d, and e).

Discussion
Resistance to DOX treatment and its adverse effects remain the major problems of its use [30].Therefore, decreasing its resistance and adverse effects, without limitation to its efficacy is recommended.Therefore, the present study aimed to investigate the effect of combining COL and DOX on the antitumor efficacy and side effects.This study revealed that EAC-harboring mice exhibited a notable elevation in the final BW. relative to control mice, which could be due to the rapid proliferation of the tumor cells inside the abdominal cavity of the mice.Treatment with DOX decreased the BW in the EAC-harboring mice group relative to untreated EAC-harboring mice.DOX high even led to a lower final BW than the control mice group, which could be attributed to DOX [31] efficacy and toxicity as an anticancer therapy [2,31].COL decreased the BW in EAC-bearing mice, reflecting its antitumor effect, as previously shown by [32].Consequently, the combination of DOX high or DOX low with COL in EAC-harboring mice led to a decrease in BW., a decrease in tumor volume, and a decrease in tumor count relative to the untreated EAC-harboring mice group.Eradication of the tumor increases survival [33], as the tumor affects the functionality of vital organs [34].In frame with DOX's known efficacy as an effective chemotherapeutic drug [35], DOX prolonged the survival of EACharboring mice.Other than the DOX survival effect, also COL enhanced the survival, but only slightly, when compared to the treatment with DOX high or low .Co-treatment of DOX low with COL increased mice survival when compared to the DOX low treatment group.In parallel to the improvement in survival, the % of early and late apoptotic EAC-cells was increased in the EACharboring mice group given DOX high , COL, or DOX low or their combinations.Co-administration of COL with DOX high didn't affect the % of apoptotic EAC-cells, significantly, when compared to DOX high alone, indicating that the maximum anticancer effect was reached by the high dose.In contrast, co-administration of COL with DOX low raised the % of early and late apoptotic cells when compared to DOX low alone, supporting the hypothesis that COL enhances the apoptotic potential of DOX low .These observations are in line with the described dosedependent potential of DOX, where an increased % of the early and late apoptotic cells occurred using several breast cancer cell lines [36].Cell cycle analysis, further, showed that treatment with either DOX high or DOX low arrested EACcells at the S-phase, whereas treatment with COL arrested the EAC-cells at the G2/M phase.This is in line with the previous papers that report DOX causes cell cycle arrest at the S-phase via topoisomerase II poisoning [37] and that COL inhibits the survival of MCF-7 cells and induces stoppage of the cell cycle at the G2/M phase [38,39].The addition of COL to the high or low DOX doses increased the stoppage of the cell cycle in the G2M phase, which could explain the increase in the antitumor effect after treatment with either combination.Concerning total RBC count, hemoglobin, and hematocrit, all these blood indices decreased in the EAC-carrying mice [40] effect via myeloperoxidase enzyme and prostaglandin E2 affection, by either tumor stress/injury or DOX treatment as toxic effect whether linked to NLRP3 inflammasome pathway or not, should be further explored.This is in line with our findings that revealed that co-treatment of EAC-harboring mice with COL and DOX mitigated the inflammation burden when contrasted with EACharboring mice that received DOX only.

Conclusion
The present study displays that tumor cotreatment by either DOX high or DOX low with COL enhanced the antitumor effect of DOX and ameliorated the DOX-mediated adverse effects.

Study Strength and Recommendation
These data are useful to explore more the benefits of COL as such in addition to other chemotherapeutics to enhance the latter efficacy, decrease DOX dose, decrease multi-drug resistance to cancer as well as decrease DOX side effects.However, a previous case report showed that a patient with FMF and Hodgkin's lymphoma who received COL with prednisone, DOX as well as vincristine, and etoposide, suffered from severe side effects and had to stop COL during chemotherapy [55] which would be considered a limitation its use in the future clinical trials.

Future prospective
More COL toxicological studies and further clinical examination(s), however, based on our previously published recommendation [56] to use selective target inhibitor chemotherapeutic drugs (via nano-formulation) as targeting 20S proteasomes [57] may be a potential strategy for overcoming clinical drug resistance, as well as if we would add vitamin E or D to ameliorate any cardiovascular side effects [58, 59].

In summary
Although Doxorubicin is widely used in chemotherapeutic regimens, it has drastic side effects with declining efficacy.Therefore, we thought to use the anti-inflammatory medication Colchicine, commonly prescribed for the treatment of gout and FMF, to reduce Doxorubicin-associated toxicity and enhance its antitumor activity/efficacy.Investigation of apoptosis, cell cycle, survival, tumor count and tumor volume, and inflammatory and biochemical markers were done in an Ehrlich Ascites Carcinoma bearing mice model, which demonstrated potential promising effect(s).However, further investigations should be done to enable their combined use in clinical settings.
[46]44] may be overcome via treatment with DOX and COL.Platelet count did not differ in the EAC-carrying mice group relative to the control group.Inoculation of EAC tumor increased the total WBCs count as previously shown [41], whereas, administration with DOX high or low doses reduced the total WBC count.Treatment with COL also decreased WBCs count which is in line with a previous study[42].Inoculation of EAC cells in different mice groups caused an increase in AST, ALT activities, urea, and creatinine levels, pointing to hepatic and renal toxicities, following tumor inoculation as in accodance with the previous studies[43,44].Subjecting EAC-carrying mice to DOX, COL, or their combination diminished liver and kidney toxicities when compared to the EAC-carrying mice group.DOX low had the same effect as DOX high , but to a lesser extent.In line with its known hepatic and renal protective properties [21, 45], COL reduced the increased liver and kidney function tests (AST and ALT, urea and creatinine levels).Serum TC, TAG, and LDL-C levels were elevated in the EAC-bearing mice group relative to the control mice group, whereas HDL-C levels were decreased in EAC-harboring mice vs the control group, supporting the findings of[46].Administration with DOX high resulted in a decrease in serum TC and TAG relative to EAC-harboring mice, whereas treatment with COL or DOX low decreased TC but not TAG levels.This shows that COL or DOX low has a potential antitumor effect, but not as DOX high .However, a previous study showed that COL Administration of DOX high , further, raised the CRP and IL-1β levels when contrasted with untreated EAC-harboring mice.This may be due to the action of DOX on the NLRP3 inflammasome pathway [9].DOX low only slightly increased CRP and IL-1β levels in EAC mice indicating a dose-dependent inflammatory response.In contrast, COL-treated EAC-bearing mice exhibited lower CRP and IL-1β levels when compared to EAC-bearing mice, which can be explained by the NLRP3 inflammasome pathway-inhibitory capacity of COL [15], but platelets activation [53] or neutrophils infiltration [54] ALT, Alanine transaminase; ANOVA, Analysis of variance; ASC, Apoptosis associated Speck-like protein containing a CARD; AST, Aspartate transaminase; Bax, B-cell lymphoma-2-associated X; Bcl-2, B-cell lymphoma-2; BW, Body weight; % BW, The percentage of body weight change; CARD, Caspase Activation and Recruitment Domains, CAT, Catalase; COL, Colchicine; CRP, C-reactive protein; DOX, Doxorubicin; DOX high , High dose of doxorubicin; DOX low , Low dose of doxorubicin; EAC, Ehrlich