Combined Efficacy of Cinnamomum Zeylanicum And Doxorubicin Against Leukemia Through Regulation of TRAIL And NF-Kappa B Pathways In Rat Model

doi:10.21203/rs.3.rs-478779/v1

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Combined Efficacy of Cinnamomum Zeylanicum And Doxorubicin Against Leukemia Through Regulation of TRAIL And NF-Kappa B Pathways In Rat Model

https://doi.org/10.21203/rs.3.rs-478779/v1

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Background: Recent discoveries in cancer therapeutics have proven combination therapies more effective than individual drugs. This study describes the efficacy of Cinnamomum zeylanicum and doxorubicin combination against benzene-induced leukemia.

Methods and Results: Brine shrimp assay was used to assess the cytotoxicity of Cinnamomum zeylanicum, doxorubicin and their combination. After AML induction in Sprague Dawley rats, the same drugs were given to rat groups. Changes in organ weights, haematological profile, and hepatic enzymes were determined. Real-time PCR was used to elucidate the effect on the expression of STMN1, GAPDH, P53 and various TRAIL and NF-kappa B components. Cinnamomum zeylanicum reduced the cytotoxicity of doxorubicin. The combination treatment showed better anti-leukemic potential than any of the individual drugs as evident from STMN1 expression (p<0.001). It was particularly useful in reducing total white blood cell counts and recovering lymphocytes, monocytes and eosinophils along with hepatic enzymes ALT and AST (p<0.001). All doses recovered relative organ weights and improved blood parameters. The combination therapy was particularly effective in the inhibition of proliferation marker GAPDH (p<0.001) and NF-kappa B pathway components Rel-A (p<0.001) and Rel-B (p<0.05). Expressions of TRAIL components c-FLIP (p<0.001), TRAIL ligand (p<0.001) and caspase 8 (p<0.05) were also altered.

Conclusion: Cinnamomum zeylanicum in combination with doxorubicin helps to counter benzene-induced cellular and hepatic toxicity and improve haematological profile. The anti-leukemic effects are potentially mediated through inhibition of GAPDH and NF-kappa B pathway and regulation of TRAIL pathway. Our data suggest the use of Cinnamomum zeylanicum with doxorubicin to improve anti-leukemic therapeutic regimes.

Molecular Genetics

Molecular Biology

Cinnamomum zeylanicum

doxorubicin

acute myeloid leukemia

TRAIL

NF-kappa B

rat model

Leukemia is a malignancy of white blood cells (WBCs) with 437 thousand estimated cases per year worldwide [1]. It is known as the uncontrolled division of abnormal WBCs produced from hematopoietic stem cells in the bone marrow [2]. Acute Myeloid Leukemia (AML) is characterized by an increase in the number of myeloid cells and proliferation of leucoplasts along with decrease in red blood cells (RBCs) and platelets [3]. AML occurs mainly in adults under the age of 60 [4]. Different factors including radiation, chemical injuries, chromosomal abnormalities, and viral infections are involved in causing leukemia, but exposure of benzene is most common. Benzene induces the cancer of the myeloid cells by its secondary metabolites like phenol, catechol and hydroquinone [5]. When benzene is metabolized in liver, a large number of reactive oxygen species (ROS) are produced which causes cytotoxicity that leads to leukemia [6, 7].

Doxorubicin is a widely used chemotherapeutic drug against AML. It induces apoptosis by DNA damage through intercalation [8]. Moreover, it also inhibits the DNA repair mechanism. Doxorubicin, however, not only targets the cancer cells but also affects normal growing cells of the body leading to unavoidable side effects including ROS production, double stranded DNA breaks and apoptosis of normal cells [9]. Combinations of different chemotherapeutic drugs are being tested by the oncologists to increase the effectiveness of the therapeutic regimes with lower doses of individual drugs. Various phytochemical compounds including natural plant extracts have also been combined with chemotherapy and showed promising outcome. Cinnamomum zeylanicum is a plant with known antioxidant and anticancer potential and has been used as natural medicine since centuries [10]. In addition to anti-cancer potential, it has also exhibited anti-microbial, anti-diabetic, anti-oxidant, anti-inflammatory and wound healing properties [11]. It has also shown anti-proliferative efficacy in acute myeloid leukemia HL-60 cells [12]. The aim of present study was to evaluate the effect of Cinnamomum zeylanicum in conjugation with doxorubicin and to investigate the regulation of underlying molecular machinery in AML using a rat model.

2.1 Preparation of aqueous Cinnamon zeylanicumextract (CE)

Commercially available powder of Cinnamomum zeylanicum bark (1000mg) was dissolved in100mL distilled water by heating at 100°C for 3 hours to make a stock of 10mg/mL. The final volume was retained at 100mL through addition of distilled water to compensate evaporative loss. The solution was centrifuged for 10 minutes at 10,000 rpm and supernatant extract was filtered and stored at 4°C. Standard Cinnamon zeylanicum extract (CE) dose of 30mg/kg was administered to rats as per literature. The weight of rats in our experiment ranged from 200 to 250 grams. For preparing dose, simple unitary method was applied. For example, for a 250g rat the calculations were done as following:

1000g weighing rat needs Cinnamomum zeylanicum = 30mg

1g ---------------------------------------------------------- = 30/1000

250g ----------------------------------------------------- = 30/1000 x 250 = 7.5mg

Since the stock solution was 10mg/ml so 750uL CE stock was taken and mixed with 250uL distilled water to make up the volume to 1ml which was then administered orally to rat through feeding tube.

2.2 Cytotoxicity assay

For determination of optimum dose of drug and chemotherapy, the Brine shrimp assay was performed. Brine Shrimp eggs were incubated in sea salt solution for 24 hours under fluorescent light. Dilutions of Cinnamomum zeylanicum, doxorubicin and their combinations (1:1 v/v) were prepared. 10 brine shrimps were added in each dilution of Cinnamomum zeylanicum i.e., 7mg/mL, 3.5mg/mL, 1.75mg/mL and 0.875mg/mL. Similarly, Brine shrimps were added in different dilution of doxorubicin i.e. 2 mg/mL, 1 mg/mL, 0.5mg/mL and 0.25mg/mL in light of previous study [13]. Same dilutions were used for combination of Cinnamomum zeylanicum and doxorubicin. After 24 hours, the lethal concentration (LC₅₀) was calculated by counting number of alive vs dead shrimps. The value of LC₅₀ is inversely proportional to the toxicity of compound. The standard values for cytotoxicity are non-toxic > 1000µg/mL, weak toxic ≥ 500µg/mL to ≤ 1000 µg/mL, toxic < 500µg/mL).

2.3 Experimental design

The study was approved by the Institutional Review Board of Quaid-i-Azam University through letter No. #BEC-FBS-QAU2020-221.Twenty-five Sprague Dawley female rats were bought from the National Institute of Health (NIH), Islamabad which were distributed into five equal groups. Animals were allowed to acclimatize for a period of seven days. The animals were maintained at room temperature with 12 hours light/dark cycle and were allowed unrestricted access to water and standard rat feed at the Primate Facility of Quaid-i-Azam University. Leukemia was induced by 0.2mL of intravenous benzene (Sigma-Aldrich Cat. No. 71-43-2) injections diluted in normal saline in ratio 0.5:1for 14 alternate days. Group A (control) was control group which was given normal saline in the tail vein. Group B (Benz) was given 0.2mL benzene injections for 2 weeks. In group C (Cinn), each rat was given aqueous Cinnamomumzeylanicumextract 30mg/kg orally on alternate days for 2 weeks after completion of benzene injections as described previously [14, 15]. In group D (Doxo), each rat was given intravenous injections of doxorubicin (Actavis, Italy Cat. No. 25316-40-9)3.2mg/kg as described previously [13] for 2 weeks after completion of benzene administration. In group E (Cinn + Doxo), each rat was given both Cinnamomum zeylanicum orally and doxorubicin intravenously for 2 weeks after completion of benzene injections.

2.4 Morphological study and blood parameters

After dissections of rats, slides were prepared from fresh blood for morphological analysis. Slides were air dried, stained with 100 % Giemsa for 5 minutes, washed with water and then dried for microscopic analysis. AML induction was confirmed by morphological changes in blood smear indicating increase in immature blast cell numbers, less cytoplasm to nucleus ratio in WBCs and erythrophagocytosis. Blood CP (complete picture) for total cell counts was done on an automated instrument in National Agricultural Research Centre (NARC), Islamabad. Weights of the organs were measured after dissection using laboratory balance. Alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) analysis was done using biochemical diagnostic kits (Sigma-Aldrich Cat. No. AP0100, Cat. No. MAK052 and Cat. No. MAK055, respectively).

2.5 Real time PCR analysis

For relative mRNA expression analysis, RNA was extracted from freshly resected liver of rats using protocol described by Chomczynski [16]. cDNA was synthesized from mRNA using VIVANTIS kit (Cat. No. cDSK01-050). For relative gene expression MIC qPCR by Bio Molecular Sciences (BMS) was used. Beta-actin was used as the housekeeping gene. Sequences of the primers used for screened genes were:

STMN1 (F-TTGCCAGTGGATTGTGTAGAG, R-TTCTTTTGATCGAGGGCTGAG),

GAPDH (F-TCCAGTATGACTCTACCCACG, R-CACGACATACTCAGCACCAG),

P53 (F-TCCGACTATACCACTATCCACTAC, R-GCACAAACACGAACCTCAAAG),

Rel-A (F-CTACGAGACCTTCAAGAGCATC, R-GATGTTGAAAAGGCATAGGGC),

Rel-B (F-CTTTTCTCAAGCTGACGTGC, R-AGATCTCCAGGTCCTCGTATG),

c-FLIP (F-AGAAGCCCTCACCTTGTTTC, R-CTCTTGTCCTTGGCTACCTTG),

DR5 (F-TCAACCCTGTGCCAATCC, R-ATGAACTCCTTCCAGCGTG),

TRAIL ligand (F-CACATTACCGGGATCACTCG, R-AGCTCTCCGTTTCTCAAGTG),

Caspase 8 (F-GAGCTGCCAGTTTCTGTTTTG, R-GTTGAAGATCAGACAGTACCCC).

2.6 Data and statistical analysis

Relative Expression Software Tool (REST-384, version 2) was used for the relative gene expression. Descriptive statistics were computed using Graph Pad Prism8software and results were described as mean ± standard deviation (SD). Inter-group comparisons were made using one-way analysis of variance (ANOVA). Groups were further separated using Tukey’s post-hoc test. Statistical difference (< 0.05 = *, < 0.01 = **, < 0.001 = ***) with a p-value < 0.05 was taken as significant and p < 0.001 was marked highly significant.

3.1 Cinnamomum zeylanicum reduced the cytotoxicity of doxorubicin.

The cytotoxicity assay results revealed that Cinnamomum zeylanicum was non-toxic (LC₅₀ = 38980µg/mL) while doxorubicin posed significant toxicity (LC₅₀ = 228.5µg/mL) to the Brine shrimps. However, using the combination of doxorubicin with Cinnamomum zeylanicum decreased the cytotoxicity of doxorubicin to LC₅₀ value of 11694.1µg/mL rendering it non-toxic. Generally, compounds with LC₅₀ of > 1000 µg/mL are considered as non-toxic, those having LC₅₀ between 1000 µg/mL to 500 µg/mL are weakly toxic and those having LC₅₀ of < 500 µg/mL are considered toxic. In this way, we can comfortably say that Cinnamomum zeylanicum masked the toxicity of doxorubicin.

3.2 Recovery of relative organ weights in treated groups.

The relative organ weight of heart, kidney and liver was substantially increased in leukemic rats after benzene administration potentially due to the hypertrophy of tissues. The treatment groups showed significant decline (p<0.05) in the organ’s weights. Cinnamomum zeylanicum alone was as effective as the combination therapy in reducing heart and kidney weights. For liver, the combination treatment showed lowest (p<0.01) reduction in weight as shown in Fig. 1.

3.3 Restoration of blood cell counts and morphological features.

The level of WBCs was significantly increased while the level of RBCs and platelets were remarkably reduced in leukemic rats (p < 0.05) as depicted in Fig. 2. The WBC counts were significantly improved by combination of doxorubicin and Cinnamomum zeylanicum (p < 0.05). Levels of RBCs and platelets were significantly recovered in all treatment groups (p < 0.001). Hemoglobin levels were significantly decreased upon benzene administration but were not affected by any of the treatment regimens (p > 0.05). Morphological analysis exhibited decreased concentration of erythrocytes and presence of abnormal leucocytes in benzene administered leukemic rats. Increased nucleus-to-cytoplasm ratio and leucocytosis was observed. However, treatment with combination therapy restored the normal morphology of leukocytes in treated rats (Fig. 2E).

3.4 Immunomodulatory effects of combination therapy

The populations of neutrophils, monocytes and eosinophils were significantly increased while the lymphocytes were significantly decreased in response to benzene administration as shown in Fig. 3. The combination of Cinnamomum zeylanicum and doxorubicin was most effective (p < 0.05) in recovering lymphocyte, monocyte and eosinophil counts. Neutrophil counts were not recovered by any of the treatments. Eosinophil population was improved by all treatment regimens (p < 0.05).

3.5 Recovery of hepatic enzyme levels upon treatment

Hepatic biomarkers ALT and AST showed marked increase while ALP showed substantial decrease upon leukemia induction in rats (p < 0.05). It was observed that combination therapy was most effective for reducing ALT and AST levels (p < 0.05). ALP levels however, were improved by individual drugs. The results are shown in Fig. 4.

3.6 Confirmation of Acute Myeloid Leukemia by STMN1 expression

The genetic marker STMN1 was used for the confirmation of acute myeloid leukemia induction.^17,18 STMN1 expression increased significantly upon benzene treatment confirming leukemia in our rats (p < 0.05). Cinnamomum zeylanicum treatment did not have any marked effect on its expression. Doxorubicin, being the classical chemotherapy, significantly reduced the expression (p < 0.001). However, the combination therapy was the most effective in lowering the STMN1 expression close to normal levels (p < 0.001) signifying the importance of our therapeutic regime as shown in Fig. 5A.

3.7 Restoration of GAPDH and P53 gene expression

GAPDH is a marker of increased metabolism and proliferation in transformed cells. Its expression was significantly increased in the benzene group (p < 0.001). All the treatment groups significantly lowered its levels (p < 0.001) but the most significant reduction was seen in the combination group (Fig. 5B). The expression of tumor suppressor gene p53 was significantly decreased upon leukemia induction (Fig. 5C). The levels improved significantly upon treatment with Cinnamomum zeylanicum alone (p < 0.001). However, the combination did not prove to be effective potentially due to masking of Cinnamomum zeylanicum effects by chemotherapy.

3.8 Inhibition of canonical and non-canonical NF-kappa B pathways

NF-kappa B pathway is involved in the proliferation of cancer cells. The expression of NF-kappa B genes Rel-A (from canonical pathway) and Rel-B (from non-canonical pathway) was increased (p < 0.05) in response to benzene induced leukemia depicting higher proliferation levels (Fig. 5D-E). Cinnamomum zeylanicum alone and the combination therapy significantly reduced the expression of both Rel-A and Rel-B genes, thus showing the anti-proliferative potential (p < 0.05).

3.9 Regulation of TRAIL pathway by various treatment regimes

TRAIL is one of the important pathways which induces apoptosis in transformed cell and can be regulated by intrinsic and extrinsic pathways. Cinnamomum zeylanicum and combination therapy showed promising results in modulating TRAIL pathway. The expression of c-FLIP, which is an inhibitor of apoptosis, was increased in response to leukemia induction but most effectively reduced by the combination of Cinnamomum zeylanicum with doxorubicin (p < 0.001) as given in Fig. 6A.DR5 expression which is known to induce apoptosis was increased by Cinnamomum zeylanicum (p < 0.01); however, its combination with doxorubicin diminished its effects (Fig. 6B). TRAIL ligand expression was increased by benzene (p < 0.001) but the combination therapy reduced the levels back to normal (Fig. 6C). In addition, the combination therapy significantly increased the expression of caspase 8 (p < 0.05) which plays a central role in extrinsic TRAIL pathway (Fig. 6D). Cinnamomum zeylanicum also showed some improvement in the expression levels of Bax, caspase 7 and 9 but did not show any effects in combination with doxorubicin (data not significant).

Leukemia is a hematopoietic malignancy of non-epithelial cells [19] that is divided into four types including AML which could also be artificially induced through benzene exposure [20]. The most convenient way to treat leukemia is chemotherapy [21]. However, due to the cytotoxicity induced by the chemotherapeutic drugs in normal cells, most patients suffer badly during treatment [22]. Currently, more focus has been shifted towards herbal medicines extracted from medicinal plants to augment chemotherapy [11]. Cinnamomum zeylanicum being a source of several beneficial compounds has proven to be an important herbal drug against leukemia. Cinnamaldehyde, one of its active ingredients, induces apoptosis in human leukemic cells through reactive oxygen species (ROS) generation leading to loss of mitochondrial membrane potential, thereby releasing cytochrome c into their cytosol. Evidence also suggests involvement of mitochondria-dependent cytochrome c-Apaf-1-procaspase-9 pathway in cinnamaldehyde induced apoptosis [23]. Cinnamaldehyde is also a potent chemotherapeutic sensitizer, as it inhibits STAT3 and AKT pathways signaling to overcome drug resistance in chemo-resistant cancer cells [24]. Likewise, eugenol, which accounts for 2.77% of cinnamon bark suppresses Nuclear Factor – Kappa B activation pathway in acute myeloid leukemia resulting in cancer cell growth arrest [25]. It also targets Notch-Hes1 signaling pathway to eliminate resistant ovarian cancer stem cells [26]. This study revealed the anticancer efficacy of combination treatment of Cinnamomum zeylanicum bark aqueous extract with doxorubicin in benzene administered leukemic rats.

Cytotoxicity assay revealed non-toxic nature of Cinnamomum zeylanicum as reported previously [12, 15]. Our data suggest that the toxicity of doxorubicin was reduced by its combination with Cinnamomum zeylanicum which is in line with previous literature [27, 28] The induction of AML was confirmed by using STMN1 genetic marker (Fig. 5A) [29] as well as morphological changes in blood smear (Fig. 2E) [30].

The increase in organ weights during in-vivo experimentation indicated increased proliferation of cells leading to hypertrophy in these organs (Fig. 1A, B, C). Hypertrophy observed is due to infiltration of malignant leukemic cells into the liver, spleen, and other vital organs. Therefore, these organs may enlarge and increase in weight causing hypertrophy in leukemia [31]. However, the reduction of organ weights back to normal by the treatment regimens provides significant evidence of protective effect of Cinnamomum zeylanicum against benzene induced hypertrophy.

Improvement in differential WBC counts (Fig. 2A) by Cinnamomum zeylanicum and its combination with chemotherapy may be attributed to the immunomodulatory effects of Cinnamomum zeylanicum [32]. Decrease in RBC and platelet counts observed in case of leukemia was reverted to normal in treated groups. The reason may be that Cinnamomum zeylanicum has a potential to stimulate the process of protein synthesis and thus helps in the recovery of normal morphology of erythrocytes. The active ingredients in cinnamon are known to elevate the levels of thyroid hormones in animals, which play direct role in increasing metabolic rate of body, as well as indirect role in controlling the blood cells production in bone marrow, thymus and other organs through increased protein synthesis. This leads to induced erythropoiesis causing increment of red blood cells [33].

When given along with chemotherapy, Cinnamomum zeylanicum counters the toxicity posed by the drug and shows renal and hepato-protective potential [34, 35]. In leukemic rats, benzene deregulates the levels of ALP, ALT and AST due to toxicity of ROS produced by its metabolites (Fig. 4) [36]. Normally these enzymes help in detoxification of compounds. Dysregulation in their levels indicate toxicity of liver [37]. This dysregulation is significantly minimized by Cinnamomum zeylanicum treatment probably due to antioxidant, free radical-scavenging properties and reduction in lipid peroxidation [32].

STMN1 being the marker of AML showed elevated expressions (Fig. 5A) in case of benzene administration [29]. This effect of benzene was remarkably minimized by the combination therapy. The upregulation of STMN1 expression promote cell proliferation, invasion and migration [17, 18].

The expression of GAPDH was significantly reduced by all the treatment groups (Fig. 5B) suggesting the decrease in the rate of glycolysis by cancer cells which suggests less proliferation of the transformed cells [38, 39]. Expression of P53 was enhanced by Cinnamomum zeylanicum which was downregulated in case of leukemia (Fig. 5C). Loss of p53 is the common genetic alteration mostly observed in cancers [40, 41]. Doxorubicin causes apoptosis through p53 dependent or p53 independent pathway by ROS production [42, 43]. Our results suggested that Cinnamomum zeylanicum alone has a potential to enhance the expression of this critical tumor suppressor gene and has ability to counter cellular damage; however, the combination did not show any promise in this regard. Cinnamomum zeylanicum and combination therapy inhibit the NF-kappa B pathway by downregulation of canonical pathway component Rel-A (Fig. 5D) and non-canonical pathway component Rel-B (Fig. 5E) which suggests that along with the apoptosis inducing capability, Cinnamomum zeylanicum also has anti-proliferative potential [44].

Our results predicted that Cinnamomum zeylanicum exerted anti-cancer activity potentially through its metabolites. Its combination with doxorubicin modulates the TRAIL pathway by increasing the expression of DR5 (Fig. 6B) and decreasing the expression of inhibitory protein c-FLIP (Fig. 6A). Doxorubicin is known to induce apoptosis through p53 dependent and independent pathways [45]. At low dose, doxorubicin displayed promising results in increasing DR5 and decreasing c-FLIP expression in human prostate cell lines [46, 47]. It is pertinent to mention that although TRAIL is known to induce apoptosis however, high expression of TRAIL is usually seen in transformed cells (Fig. 6C) [48]. TRAIL expression is increased as a first line of defense against tumorigenesis. However, its presence does not necessarily induce apoptosis as numerous other players are also involved in the process including the death and decoy receptors and the presence or absence of inhibitory proteins [48]. The combination therapy was highly effective in increasing caspase 8 expression in our study (Fig. 6D). Cinnamomum zeylanicum has already shown promising efficacy in inducing caspase-dependent apoptosis through its metabolites [49] e.g., cinnamaldehyde had shown apoptotic effect by increasing pro-apoptotic protein Bax and P53 expression on liver cancer cell [50]. Thus, our results clearly indicate the modulation of apoptosis through TRAIL pathway by combination therapy.

We conclude that Cinnamomum zeylanicum aqueous extract increases the anti-cancer and therapeutic potential of doxorubicin. It also shows remarkable ability of decreasing the cytotoxicity caused by doxorubicin. The combination treatment exhibits excellent anti-leukemic potential and improves the blood parameters, reduces organ hypertrophy and restores the hepatic enzyme levels. The anticancer potential is mediated through GAPDH, TRAIL and NF-kappa B pathways. Hence Cinnamomum zeylanicum extract can be administered/combined with doxorubicin as an excellent chemotherapeutic agent against AML.

Acknowledgements

We acknowledge the technical assistance provided by Alpha Genomics in sample processing, National Training and Research Laboratory for providing cDNA synthesis kits, National Institute of Health for providing rats and NARC for facilitation in hematological profiling.

Funding

The work was financially supported by University Research Fund (URF) of Quaid-i-Azam University, Islamabad, Pakistan

Disclosure/Conflict of Interest

All authors declare no conflict of interests.

Availability of Data and Material

All data recorded during experiments is made available in the manuscript in transparent manner.

Code availability

Not Applicable

Authors Contribution

Conceptualization: Anees M, Naeem A, Methodology: Naeem A, Khan IU, Essa A, Software: Bukhari S, Siddique MH, Validation: Formal analysis: Sultan A, Murtaza I, Anis RA, Investigation: Naeem A, Khan IU, Data curation: Siddique MH, Essa A, Ali Z, Original draft preparation: Anees M, Bukhari S, Review and editing: Anees M, Bukhari S, Vanhara P, Moran L

Ethics Approval

The study was approved by the Institutional Review Board of Quaid-i-Azam University through letter No. #BEC-FBS-QAU2020-221 and undertaken after getting ethical approval from Quaid-i-Azam University, Islamabad, Pakistan

Consent to Participate

All authors submitted their consent to participate in experiments performed, analysed and displayed in manuscript

Consent for Publication

All authors submit their consent for publication of this manuscript in Molecular Biology Reports Journal.

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Editorial decision: Major Revisions Needed
25 May, 2021
Reviewers invited by journal
03 May, 2021
Reviews received at journal
03 May, 2021
Editor assigned by journal
01 May, 2021
First submitted to journal
28 Apr, 2021

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Combined Efficacy of Cinnamomum Zeylanicum And Doxorubicin Against Leukemia Through Regulation of TRAIL And NF-Kappa B Pathways In Rat Model

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