Ocimum basilicum L. (basil) presents pro-apoptotic activity in an Ehrlich’s experimental tumor murine model

ABSTRACT

Purpose:

This study aimed to evaluate the therapeutic effect of an ethanol extract of Ocimum basilicum L. (EEOb) aerial parts against Ehrlich’s experimental tumor (EET) in mice.

Methods:

Swiss mice were divided into two groups (control and treated; n = 6). On day 21, all mice were inoculated subcutaneously with 2 × 10⁶ (0.05 mL) EET cells in the left paw for solid tumor development. This study lasted 28 days. Treatment began 24 hours after inoculation with EET. Measurements of dorsoplantar thickness were used to assess tumor growth. The paw pad was collected for histopathological analysis and stained using the argyrophilic nucleolar organizing regions (AgNOR) technique and immunohistochemistry for proliferating cell nuclear antigen, Bcl-2 and Bax.

Results:

The treatment of animals with EEOb at 100 mg/kg intraperitoneally was able to reduce the growth (Control = 3.7 ± 0.1 mm vs. EEOb = 5.7 ± 0.2 mm) and the number of AgNORs of solid Ehrlich tumor. The antitumor effect of EEOb was associated with the induction of apoptosis of tumoral cell, as suggested by the reduction of the content of Bcl-2 induced by extract.

Conclusions:

The study demonstrated that daily administration of EEOb is able to reduce the growth of EET by induce apoptosis of tumoral cells.

Key words
Ocimum basilicum ; Apoptosis; Breast Neoplasms

Introduction

Ocimum basilicum L. (basil) (Lamiaceae) is an aromatic spice with a unique flavor whose leaves, dried or fresh, are commonly used in culinary applications worldwide^1,2. In addition to culinary applications, basil also has been widely used in folk medicine to treat a wide range of diseases such as headache, cough, diarrhea, dyspepsia, flatulence, gastritis, renal disfunction (as a diuretic agent), and diabetes³. In this regard, many studies have confirmed numerous pharmacological properties of O. basilicum by pre-clinical and clinical trials^4–6.

In-vitro studies have demonstrated that ethanol extracts from aerial parts of O. basilicum are active against several humans tumor cells such as cervical cancer (HeLa)^6,7, breast cancer (MCF-7, T47D and MDA-MB-231)^6–8, acute T cell leukemia (Jurkat)⁷, erythroleukemia (K562)⁶, colorectal adenocarcinoma (HT-29 and HCT₁₁₆)^7,9, pancreatic cancer (MIAPaCa-2)⁷, prostate cancer (PC3)⁶, and melanoma (B₁₆F₁₀ cells)¹⁰. The antitumoral effects were attributed to the phenolic flavonoids and tannins compounds frequently found in O. basilicum extracts^6,7,9,11.

However, despite the dense body of evidence related to the antitumor activity of O. basilicum in vitro, little is known about the effect of this medicinal plant in solid tumor models in vivo^11,12. Ehrlich experimental tumor (EET) is a primitive undifferentiated mice breast carcinoma that grows in several murine strains in solid form when inoculated subcutaneously¹³. It presents many similarities with human tumors and is sensitive to anticancer drugs, thus making the EET a valuable tool in exploring the effect of potential cancer chemicals and drugs^13–16. Therefore, the aim of present study was to evaluate the antitumoral activity of ethanol extract of O. basilicum L. (basil) aerial parts against EET in mice.

Methods

Preparation of ethanol extract of Ocimum basilicum L. (basil) aerial parts

Plants aerial parts of O. basilicum species were collected in Carmópolis de Minas, Minas Gerais, Brazil. An exsiccate was banked at the Herbarium of the Institute of Biological Sciences, Universidade Federal de Minas Gerais, with the register number BHCB 147240. To prepare ethanol extract, 110.8 g of fresh leaves of O. basicilum was dissolved in 1,000-mL ethanol 70% v/v in laboratory condition for 10 days. This material was filtered and dried using a rotary evaporator (Fisatom 803; Uberlândia, MG, Brazil), and the yield of these procedure was 5,56 g of ethanol extract. These dried powders were resuspended in saline (NaCl 0,9%) and used in the treatment group.

Animals

Six-week-old male Swiss mice (Central Vivarium of the Universidade Federal de São João del-Rei, Campus Dom Bosco, São João Del-Rei, MG, Brazil), weighing 30 to 40 g, were used in this study. The animals were kept with solid chow and water ad libitum in an environment with a controlled temperature of 25 ± 2°C, 40% humidity, and a 12/12-h light/dark cycle.

All experimental procedures strictly followed the international protocols for laboratory animal management, and the methods were approved by the Laboratory Animal Research Ethics Committee of the Universidade Federal de São João del-Rei (protocol nº 033/2012).

The animals were divided into two groups (i.e., control and treated) with six animals each, housed in polycarbonate cages (40 × 45 × 25 cm) and submitted to a 10-day adaptation period prior to the start of the experiment.

Preparation and inoculation of Ehrlich experimental tumor

The EET cells were maintained in the ascitic form by weekly inoculations with 1 × 10⁶ cells in Swiss mice intraperitoneally (i.p.)¹⁷. For the experimental procedures, 3 mL of the ascitic fluid were removed, centrifuged (3 min, 2,000 g), and the supernatant was removed. The EET cells were washed three times in saline, counted in the Neubauer’s chamber, and the viability was determined by the trypan blue dye exclusion staining. On the 21st day, all mice were inoculated subcutaneously with 2 × 10⁶ (0.05 mL) EET cells in the left footpad for the development of the solid tumor¹⁸.

Treatment of animals with ethanol extract of Ocimum basilicum L.

All the animals of control and treated group received daily, through intraperitoneal injection (i.p.), 0.05 mL of saline and 0.05 mL of ethanol extract of O. basilicum L. (EEOb) at 100 mg/kg, respectively, until the end of the experiment. These study lasted 28 days. The treatment started 24 h after the inoculation of EET.

Tumor growth evaluation

For the tumor growth curve evaluation, measurements (duplicate) of the left hind limb dorsal-plantar thickness were carried out. This measurement was performed with a digital caliper immediately before and after the inoculation of the tumor, three times a week, for 28 days. The mean values obtained were considered as the value of the tumor growth of the day¹⁹.

Histochemical

Animals were euthanized with anesthesia overdose (100 mg/kg ketamine + 12 mg/kg xylazine, i.p.), and the left footpad was collected for histopathological analysis. Tissues samples were fixed in neutral 10% buffered formalin (pH 7.2) at room temperature. After fixation, tissues were dehydrated through graded alcohol solutions, and embedded in paraffin. Sections (4-μm thickness) were stained using the argyrophilic nucleolar organizer regions (AgNOR) technique^20–22. Next, AgNORs were counted using a digital image analyzer (KS300 Program; version 2.0, Kontron Elektronik; London, United Kingdom) from images generated by a Zeiss Axiolab microscope connected to a camera interconnected to a board computer scanner. A total of 30 cell nucleus per case, selected at random in slide, were analyzed at a 1,000x magnification.

Immunohistochemistry

To evaluate the proliferative activity of the cells, immunohistochemistry for proliferating cell nuclear antigen (PCNA) was performed. In order to evaluate the balance between pro and anti-apoptotic factors in the tissue, Bcl-2 and Bax were marked using the streptavidin–biotin-peroxidase method²³, allowing insights into the mechanisms related to the survival and resistance of cancer cells to treatment. Tissue sections (4-µm thick) were fixed in formalin and embedded in paraffin and mounted on poly-l-lysine-coated microscope slides. Tumor sections were deparaffinized and rehydrated through xylene and graded alcohols. After antigen retrieval, endogenous peroxidase was blocked (30 min to PCNA and 10 min to Bax/Blc-2) with 3% (v/v) hydrogen peroxide in darkroom and washed in phosphate-buffered saline (PBS). Sections were incubated overnight (4° C) with primary anti-Blc-2 (Santa Cruz Biotechnology, Santa Cruz, CA, United States of America; 1:50), anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA, United States of America; 1:200), and anti-PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, United States of America; 1:400) antibodies diluted in PBS plus bovine serum albumin (PBS-BSA). The slides were then incubated with secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA United States of America; 1:200) associated with dextran polymer by 30 min in room temperature. After washing, the slides were incubated with avidin-biotin-horseradish peroxidase conjugate (Strep ABC complex by Vectastain® ABC reagent and peroxidase substrate solution) for 30 min. Both biomarkers ware visualized with the chromogen 3,3’-diaminobenzidine (DAB). Slides were counterstained with Harry’s hematoxylin, dehydrated in a graded alcohol series, cleared in xylene, and coverslipped.

The images of the slides were acquired using an optical microscope and the Axion Vision software LE 4.8.2.0 (London, United Kingdom). For PCNA detection, photographs were recorded at 400x magnification, and the labeling frequency was determined by the percentage of positive cells in 1,000 tumor cells²⁴. For analysis of Bax and Bcl-2, ten photos were recorded, and a limit of 10% of positive tumor cells with distinct cytoplasmic pattern was considered positive for Bcl-2 and Bax. The criteria for interpreting Bcl-2 and Bax were:

• Negative score (0): 0%;

• Score 1: between 1–10% positive cells;

• Score 2: between 10–50% positive cells;

• Score 3: more than 50% of positive cells^25,26.

Statistical analysis

Data normality was assessed by the Shapiro-Wilk’s test. Statistical procedures were performed using the GraphPad Prism 5 (GraphPad Software, CA, United States of America) and the unpaired Mann-Whitney’s test, with a 5%-significance level. Data were expressed as the mean ± standard deviation.

Results and Discussion

Ocimum basilicum is a plant used in folk medicine due to its numerous therapeutic properties. Among them, its antitumor effect stands out³. Pre-clinical studies have already shown that extracts and fractions obtained from the leaves and other aerial parts of O. basilicum have recognized antiproliferative and pro-apoptotic activity against numerous human cell types of solid and hematological cancer^6–8,11. However, the scope of evidence regarding the in-vivo antitumoral effect of this medicinal plant is very scarce. Thus, in this study we aimed to evaluate the antitumoral activity of EEOb in vivo using a solid Ehrlich tumor murine model.

As showed in Fig. 1a, the intraperitoneal use of EEOb at 100 mg/kg significantly reduces the tumor growth in animals (3.7 ± 0.1 mm) compared to the untreated group (5.7 ± 0.2 mm; p < 0.05). For animals that received EEOb, the footpad thickness was significantly reduced from the sixth day to the end of the 28th day of the experiment. Similarly, other studies showed that the oral use of 50% alcoholic aqueous extract of O. basilicum (200 mg/kg, p.o.) resulted in significant reduction in tumor volume and lethality in a murine melanoma model (B₁₆F₁₀ cells)¹¹.

Antitumoral activity of O. basilicum can be attributed to some compounds such as polyphenols, hydroxycinnamic acids (caffeic acid, coumarin acid, and rosmarinic acid), flavonoids, and volatile compounds (terpenoids, carotenoids, phenylpropanoids/benzenoids, fatty acid derivatives, and C5-branched compounds)^27–29. The chemical composition of volatile compounds of EEOb employed in this study was investigated previously by Araújo et al.³⁰ using gas chromatography/mass spectrometry (GC/MS). The major compounds of EEOb were 9,12,15-octadecatrienoic acid ethyl ester (26%), phytol (18%), and cadinene (11%). The phytol has inhibitory effects on the pro-carcinogen factors, and induced genotoxicity and apoptosis in tumoral cells of Swiss mice with DMBA-induced breast cancer³¹.

The essential oil extracted from wood of Phoebe bournei (Hemsl.) Yang, which is rich in δ-cadinene, has good antitumor activity against leukemia (HL-60; IC₅₀ 42.7 µg/mL), hepatoma (SMMC-7721; IC₅₀ 88.1 µg/mL), breast cancer (MCF-7; IC₅₀ 54.9 µg/mL), and colon carcinoma (SW480; IC₅₀ 75.6 µg/mL) human cell types³². These findings help to explain the antitumoral activity of EEOb in vivo against ETT showed in this study. Furthermore, the numerous phytochemical constituents in EEOb and their potency to prevent cancer cell growth may be attributed to the additive or synergistic effects of the different components.

The number of AgNOR is related to the cell cycle and proliferative activity of the cell³³. The quantification of AgNOR area is related to the tumor growth rate, cell proliferative activity and consequently tumor progression^34,35. Thus, we used AgNOR quantification to evaluate the antitumor effect of EEOb in vivo. In accordance with Fig. 1, the AgNOR number (Control = 2.40 ± 0.31 vs. EEOb = 1.60 ± 0.15; p < 0.05) (Figs. 1b and 1c) per nucleus morphometric analysis was significantly higher in the control group compared to animals treated with EEOb (100 mg/Kg). In squamous cell carcinoma of the tongue, there is a correlation between the amount of AgNORs and the degree of differentiation, proliferation rate, and malignancy of the tumor³⁶. This correlation was also seen in oral squamous cell carcinoma^37,38. Thus, the reduction in the number of AgNORs induced by EEOb suggests that the treatment decreases the rate of cellular proliferation of the EET and impairs its malignity.

Figure 1
Antitumoral activity of ethanol extract of Ocimum basilicum L. (basil) (EEOb) aerial parts against Ehrlich experimental tumor in vivo. (a) Tumor growth curve in mice footpads for 28 days. (b) Photomicrographs show the subcutaneous tissue of the implantation the Ehrlich tumor stained using the argyrophilic nucleolar organizer regions (AgNOR) technique (1,000X magnification). (c) Number the AgNORs of mice footpads inoculated with EET after 28 days. Control group received daily saline solution. EEOb group received daily basil extract at 100 mg/kg intraperitoneally. Data are mean ± standard deviation (n = 6).

In order to evaluate the possible mechanism of antitumor action of the EEOb, immunohistochemistry for markers of cell proliferation (PCNA) and apoptosis (Bcl-2 and Bax) was performed. Figure 2 shows that the treatment of animals with EEOb at 100 mg/kg was not able to reduce the expression of PCNA (Control = 40.95 ± 6.40% vs. EEOb = 55.64 ± 6.88%; p = 0.064) and Bax (Control = 24.50 ± 4.95% vs. EEOb = 22.10 ± 4.33%; p = 0.841) from EET. However, the Bcl-2 content is significantly reduced after treatment with the extract (12.48 ± 3.20%) compared to untreated animals (22.57 ± 4.75%; p = 0.016). These results shows that EEOb induces apoptosis in EET cells by reducing the content of the anti-apoptotic protein Bcl-2.

Figure 2
Immunohistochemistry of (a) proliferating cell nuclear antigen (PCNA), (b) Bax and (c) Bcl-2 in mice footpads inoculated with Ehrlich experimental tumor after 28 days and treated with ethanol extract of Ocimum basilicum L. (basil) (EEOb) aerial parts. Control group received daily saline solution. EEOb group received daily basil extract at 100 mg/kg intraperitoneally. Data are mean ± standard deviation (n = 6).

Corroborating this study, it was previously demonstrated that the ethanolic extract of O. basilicum decreases the expression of the Bcl-2 protein in breast cancer cells (T47D and MCF-7) in vitro at concentrations of 199 and 388 µg/ mL, respectively³⁹. Moreover, O. basilicum extract reverts the increase of Bcl-2 content from testicular tissue induced by acute exposure of cadmium in albino rats, suggesting that the effect of Basil extract on Bcl-2 is maintained in tissues other than breast tissue.

Conclusion

The results showed that the daily administration of 100 mg/kg of EEOb aerial parts was able to reduce the proliferative speed of EET, evidenced by the reduction of tumor growth and by the smaller number of AgNORs in the tumor cell nuclei.

Furthermore, we showed that this effect may be associated with the ability of O. basilicum extract to induce Bcl-2-dependent apoptosis in tumor cells. However, for a more comprehensive understanding of the antitumor mechanism and related effects, further studies are needed to investigate in more detail the cellular signaling pathways involved, the interaction of the extract with other apoptotic proteins, as well as preclinical and clinical studies to evaluate its efficacy and safety in animal and human models. These additional research efforts may provide essential insights for the development of O. basilicum-based therapies for cancer treatment.

Acknowledgements

The authors are grateful to Universidade Federal de São João del-Rei by the support and to prof. dr. Alexandre Salino for the plant material identification.

• Research performed at Laboratory Experimental Pathology of the Department of Medicine of the Universidade Federal de São João del-Rei, Divinópolis, MG, Brazil.

• Funding

  Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
  Finance Code 001
  Fundação de Amparo à Pesquisa do Estado de Minas Gerais
  Grants No: PPM-00229-16, APQ-00068-18

Data availability statement

Data will be available upon request.

References

• ¹
  Prinsi B, Morgutti S, Negrini N, Faoro F, Espen L. Insight into Composition of Bioactive Phenolic Compounds in Leaves and Flowers of Green and Purple Basil. Plants (Basel). 2019;9(1):22. https://doi.org/10.3390/plants9010022
  » https://doi.org/10.3390/plants9010022
• ²
  Vieira MC, Carlesso A, Heredia ZNA, Gonçalves WLF, Tabaldi LA, Melgarejo E. Consórcio de manjericão (Ocimum basilicum L.) e alface sob dois arranjos de plantas. Rev Bras Plantas Med. 2012;14:169–74. https://doi.org/10.1590/S1516-05722012000500008
  » https://doi.org/10.1590/S1516-05722012000500008
• ³
  Sestili P, Ismail T, Calcabrini C, Guescini M, Catanzaro E, Turrini E, Layla A, Akhtar S, Fimognari C. The potential effects of Ocimum basilicum on health: a review of pharmacological and toxicological studies. Expert Opin Drug Metab Toxicol. 2018;14(7):679–92. https://doi.org/10.1080/17425255.2018.1484450
  » https://doi.org/10.1080/17425255.2018.1484450
• ⁴
  Bozin B, Mimica-Dukic N, Simin N, Anackov G. Characterization of the volatile composition of essential oils of some lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J Agric Food Chem. 2006;54(5):1822–8. https://doi.org/10.1021/JF051922U
  » https://doi.org/10.1021/JF051922U
• ⁵
  Samson J, Sheeladevi R, Ravindran R. Oxidative stress in brain and antioxidant activity of Ocimum sanctum in noise exposure. Neurotoxicology. 2007;28(3):679–85. https://doi.org/10.1016/j.neuro.2007.02.011
  » https://doi.org/10.1016/j.neuro.2007.02.011
• ⁶
  Rezzoug M, Bakchiche B, Gherib A, Roberta A, Flamini G, Kilinçarslan Ö, Mammadov R, Bardaweel SK. Chemical composition and bioactivity of essential oils and Ethanolic extracts of Ocimum basilicum L. and Thymus algeriensis Boiss. & Reut. from the Algerian Saharan Atlas. BMC Complement Altern Med. 2019;19(1):146. https://doi.org/10.1186/S12906-019-2556-Y
  » https://doi.org/10.1186/S12906-019-2556-Y
• ⁷
  Elansary HO, Mahmoud EA. In vitro antioxidant and antiproliferative activities of six international basil cultivars. Nat Prod Res. 2015;29(22):2149–54. https://doi.org/10.1080/14786419.2014.995653
  » https://doi.org/10.1080/14786419.2014.995653
• ⁸
  Al-Ali KH, El-Beshbishy HA, El-Badry AA, Alkhalaf M. Cytotoxic activity of methanolic extract of Mentha longifolia and Ocimum basilicum against human breast cancer. Pak J Biol Sci. 2013;16(23):1744–50. https://doi.org/10.3923/PJBS.2013.1744.1750
  » https://doi.org/10.3923/PJBS.2013.1744.1750
• ⁹
  Abdelhady MIS, Motaal AA. A cytotoxic C-glycosylated derivative of apigenin from the leaves of Ocimum basilicum var. thyrsiflorum Rev Bras Farmacogn. 2016;26(6):763–6. https://doi.org/10.1016/J.BJP.2016.06.004
  » https://doi.org/10.1016/J.BJP.2016.06.004
• ¹⁰
  Ahmed AF, Attia FA, Liu Z, Li C, Wei J, Kang W. Antioxidant activity and total phenolic content of essential oils and extracts of sweet basil (Ocimum basilicum L.) plants. Food Sci Hum Wellness. 2019;8(3):299–305. https://doi.org/10.1016/j.fshw.2019.07.004
  » https://doi.org/10.1016/j.fshw.2019.07.004
• ¹¹
  Monga J, Sharma M, Tailor N, Ganesh N. Antimelanoma and radioprotective activity of alcoholic aqueous extract of different species of Ocimum in C(57)BL mice. Pharm Biol. 2011;49(4):428–36. https://doi.org/10.3109/13880209.2010.521513
  » https://doi.org/10.3109/13880209.2010.521513
• ¹²
  Mahmoud GI. Biological effects, antioxidant and anticancer activities of marigold and basil essential oils. J Med Plantas Res. 2013;7(10):561–72.
• ¹³
  Feitosa IB, Mori B, Teles CBG, Costa AGD. What are the immune responses during the growth of Ehrlich’s tumor in ascitic and solid form? Life Sci. 2021;264:118578. https://doi.org/10.1016/J.LFS.2020.118578
  » https://doi.org/10.1016/J.LFS.2020.118578
• ¹⁴
  Calixto-Campos C, Zarpelon AC, Corrêa M, Cardoso RDR, Pinho-Ribeiro FA, Cecchini R, Moreira EG, Crespigio J, Bernardy CCF, Casagrande R, Verri, WA, Jr. The Ehrlich tumor induces pain-like behavior in mice: a novel model of cancer pain for pathophysiological studies and pharmacological screening. Biomed Res Int. 2013;2013:624815. https://doi.org/10.1155/2013/624815
  » https://doi.org/10.1155/2013/624815
• ¹⁵
  Bahr HI, Toraih EA, Mohammed EA, Mohammad HM, Ali EA, Zaitone SA. Chemopreventive effect of leflunomide against Ehrlich’s solid tumor grown in mice: Effect on EGF and EGFR expression and tumor proliferation. Life Sci. 2015;141:193–201. https://doi.org/10.1016/J.LFS.2015.10.003
  » https://doi.org/10.1016/J.LFS.2015.10.003
• ¹⁶
  Queiroz ML, Valadares MC, Bincoletto C, Dieamant GC. Ehrlich ascites tumor as a tool in the development of compounds with immunomodulatory properties. Immunopharmacol Immunotoxicol. 2004;26(4):511–25. https://doi.org/10.1081/IPH-200042289
  » https://doi.org/10.1081/IPH-200042289
• ¹⁷
  Lisičić D, Benković V, Ðikić D, Blazevic AS, Mihaljevic J, Orsolic N, Knezevic AH. Addition of propolis to irinotecan therapy prolongs survival in ehrlich ascites tumor-bearing mice. Cancer Biother Radiopharm. 2014;29(2):62–9. https://doi.org/10.1089/CBR.2013.1535
  » https://doi.org/10.1089/CBR.2013.1535
• ¹⁸
  Pinto FC, Menezes GB, Moura SA, Cassali GD, Teixeira MM, Cara DC. Induction of apoptosis in tumor cells as a mechanism of tumor growth reduction in allergic mice. Pathol Res Pract. 2009;205(8):559–67. https://doi.org/10.1016/J.PRP.2009.01.009
  » https://doi.org/10.1016/J.PRP.2009.01.009
• ¹⁹
  Morais MG, da Costa GAF, Silva AG, Ribeiro RIMA, Ferreira JMS, Duarte-Almeida JM, Pinto FCH, Lima LARS. Inhibiting the development of solid ehrlich tumor by extract of the ripe fruits of Solanum lycocarpum. In: Collins HR (Ed.). Caffeic Acid, Potential Uses and Health Benefits. Nova York: Nova Science Publisher; 2017. p. 83-96.
• ²⁰
  Bolat D, Kürüm A, Canpolat S. Morphology and quantification of sheep pineal glands at pre-pubertal, pubertal and post-pubertal periods. Anat Histol Embryol. 2018;47(4):338–45. https://doi.org/10.1111/AHE.12359
  » https://doi.org/10.1111/AHE.12359
• ²¹
  Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level. Histochem J. 1986;18(1):5–14. https://doi.org/10.1007/BF01676192
  » https://doi.org/10.1007/BF01676192
• ²²
  Pillai KR, Sujathan K, Kannan S, Abraham EK, Mathew B, Amma NS, Nair MK, Menon VP. Argyrophilic nucleolar organizer regions in the evaluation of tumour progression in the oral mucosa: correlation with tissue pathology. J Cancer Res Clin Oncol. 1994;120(12):723–6. https://doi.org/10.1007/BF01194270
  » https://doi.org/10.1007/BF01194270
• ²³
  Galvão HC, Gordón-Núñez MA, de Amorim RF, Freitas Rde A, de Souza LB. Immunohistochemical expression of protein 53, murine double minute 2, B-cell lymphoma 2, and proliferating cell nuclear antigen in odontogenic cysts and keratocystic odontogenic tumor. Indian J Dent Res. 2013;24(3):369–74. https://doi.org/10.4103/0970-9290.118019
  » https://doi.org/10.4103/0970-9290.118019
• ²⁴
  Silva AE, Santos FGA, Cassali GD. Marcadores de proliferação celular na avaliação do crescimento do tumor sólido e ascítico de Ehrlich. Arq Bras Med Vet Zootec. 2006;58(4):658–61. https://doi.org/10.1590/S0102-09352006000400030
  » https://doi.org/10.1590/S0102-09352006000400030
• ²⁵
  Callagy GM, Pharoah PD, Pinder SE, Hsu FD, Nielsen TO, Ragaz J, & Caldas C. Bcl-2 is a prognostic marker in breast cancer independently of the Nottingham Prognostic Index. Clin Cancer Res. 2006;12(8):2468–75. https://doi.org/10.1158/1078-0432.ccr-05-2719
  » https://doi.org/10.1158/1078-0432.ccr-05-2719
• ²⁶
  Zaha DC, Lazăr E. Molecular characterization of apoptosis by the immunohistochemical evaluation of Bcl-2 in breast cancer. Rom J Morphol Embryol. 2012;53(1):155–60.
• ²⁷
  Güez CM, Souza ROD, Fischer P, Leão MFDM, Duarte JA, Boligon AA, Athayde, ML, Zuravski L, de Oliveira LFS, Machado MM. Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inflammatory effects in human leukocytes cell cultures exposed to challenging agents. J Pharm Sci. 2017;53(1):15098. https://doi.org/10.1590/s2175-97902017000115098
  » https://doi.org/10.1590/s2175-97902017000115098
• ²⁸
  Islam MS, Badrul Alam M, Zahan R, Sarker GC, Chowdhury NS, Maruf Khan M, Hasan M, Saifur Rahman M, Mosaddik MA, Jesmin M, Ekramul Haque M. In vitro antioxidant and anti-neoplastic activities of Ocimum sanctum leaves in Ehrlich Ascites Carcinoma bearing mice. Int J Cancer Res. 2011;7(3):209–21. https://doi.org/10.3923/IJCR.2011.209.221
• ²⁹
  Sakr SA, Nooh HZ. Effect of Ocimum basilicum extract on cadmium-induced testicular histomorphometric and immunohistochemical alterations in albino rats. Anat Cell Biol. 2013;46(2):122–30. https://doi.org/10.5115/ACB.2013.46.2.122
  » https://doi.org/10.5115/ACB.2013.46.2.122
• ³⁰
  Araújo SG, Alves LF, Pinto MEA, Oliveira GT, Siqueira EP, Ribeiro RIMA, Ferreira JMS, Lima LARS. Volatile compounds of Lamiaceae exhibit a synergistic antibacterial activity with streptomycin. Braz J Microbiol. 2015;45(4):1341–7. https://doi.org/10.1590/s1517-83822014000400026
  » https://doi.org/10.1590/s1517-83822014000400026
• ³¹
  Alencar MVOB, Islam MT, de Lima RMT, Paz MFCJ, Dos Reis AC, da Mata AMOF, Filho JWGO, Cerqueira GS, Ferreira PMP, Sousa JMC, Mubarak MS, Melo-Cavalcante AAC. Phytol as an anticarcinogenic and antitumoral agent: An in vivo study in swiss mice with DMBA-Induced breast cancer. IUBMB Life. 2019;71(2):200–12. https://doi.org/10.1002/IUB.1952
  » https://doi.org/10.1002/IUB.1952
• ³²
  Ding W, Ning L, Xiong Y, Shi H, Wang T, An R. Essential oils extracted from Phoebe hui Cheng ex Yang: Chemical constituents, antitumor and antibacterial activities, and potential use as a species identifier. J Wood Chem Technol. 2017;37(3):201–10. https://doi.org/10.1080/02773813.2016.1271435
  » https://doi.org/10.1080/02773813.2016.1271435
• ³³
  Sowmya GV, Nahar P, Astekar M, Agarwal H, Singh MP. Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions. Biotech Histochem. 2017;92(2):115–21. https://doi.org/10.1080/10520295.2017.1283055
  » https://doi.org/10.1080/10520295.2017.1283055
• ³⁴
  Gossler VSA, Santos FAG, Azevedo AR, Gonçalves PC, Rigolo HA, Trevisan C, Masseno APB, Cardoso APMM, Papa PC, Castilho C, Giometti IC. Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus. An Acad Bras Ciênc. 2017;89(3):1719–27. https://doi.org/10.1590/0001-3765201720150611
  » https://doi.org/10.1590/0001-3765201720150611
• ³⁵
  Santos FGA, Moro L, Cassali GD, Paixão TA, Campos PP, Silva SS, Vasconcelos AC. Cell proliferation markers in the transplanted canine transmissible venereal tumor. Arq Bras Med Vet Zootec. 2011;63(6):1345–52. https://doi.org/10.1590/S0102-09352011000600010
  » https://doi.org/10.1590/S0102-09352011000600010
• ³⁶
  Oliveira MG, Lauxen IS, Martins Neto M, Rados PV, Jaeger F, Kaizer MR, Sant’ana Filho M. Tongue Squamous Cell Carcinoma: Relationship Between Argyrophilic Nucleolar Organizer Regions (AgNORS) and Histopathologic Grading. Appl Cancer Res. 2005;25(1):20–4.
• ³⁷
  Silva SO, Gatto KP, Carli JP, Souza PHC, Busin CS. Evaluation of proliferative activity in oral squamous cell carcinoma by the AgNOR staining method. Odonto. 2011;115–21.
• ³⁸
  Tomazelli KB, Modolo F, Rivero ER. Evaluation of AgNORs in Oral Potentially Malignant Lesions. J Oncol. 2015;2015:218280. https://doi.org/10.1155/2015/218280
  » https://doi.org/10.1155/2015/218280
• ³⁹
  Hidayati DN, Zahroh F, Wahyuni L, Arifin I. Decreas of BCL-2 Expression by Ethanol Extract of Ocimum basilicum L. Leaves in Breast Cancer Cells. Jurnal Profesi Medika: Jurnal Kedokteran dan Kesehatan. 2021:15(1):7–12. https://doi.org/10.33533/jpm.v15i1.2491
  » https://doi.org/10.33533/jpm.v15i1.2491

Edited by

Section editor: Norbert Nemeth https://orcid.org/0000-0002-1162-3778

Publication Dates

• Publication in this collection
  29 July 2024
• Date of issue
  2024

History

• Received
  29 Dec 2023
• Accepted
  22 May 2024