In vitro and in vivo anticancer properties of cucurbitacin isolated from Cayaponia racemosa

Abstract

Context: Cucurbitacins are a group of triterpenoids that have a cucurbitane skeleton with a wide range of biological activities.

Objectives: This study evaluated the anticancer properties of one cucurbitacin isolated from Cayaponia racemosa Cong. (Cucurbitaceae), 2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en (1), with in vitro and in vivo models.

Materials and methods: In vitro cytotoxic activity was determined with human leukemia (HL60) and normal blood cells (PBMC). Sarcoma 180 was used as in vivo model.

Results: The cucurbitacin (1) reduced the number of viable cells; however, there was no changed in the number of non-viable cells at 5 µg/mL. Selectivity towards cancer cells was suggested by the absence of activity on normal proliferating lymphocytes at the concentrations tested (IC₅₀ >25 µg/ml). Morphological analysis of compound 1-treated cells showed typical apoptotic features, such as intense deposition of granules in the cytoplasm (eosinophilia), DNA fragmentation and irregularities in the plasma membrane. In addition, the cells treated with compound 1 presented intense vacuolization and disruption of the plasma membrane. Acridine orange/Ethidium bromide staining confirmed these findings, revealing an increased number of apoptotic cells. In the Sarcoma 180 tumor model, compound 1 showed 52 and 62% of antitumor activity, either alone (25 mg/kg/day) or in association with the chemotherapeutic agent 5-FU (10 + 10 mg/kg/day), respectively. Moreover, either alone or associated with 5-FU, treatment with compound 1 caused an increase in spleen weight and morphological alterations related to immunostimulatory properties.

Conclusion: These data indicate that these naturally occurring compounds have anticancer potential.

Keywords::

• HL60
• antitumor activity
• Sarcoma 180

Introduction

Cucurbitacins are a special group of triterpenoids that have a cucurbitane skeleton, and a wide range of biological activities is associated with their chemical structural complexity (Chen et al., 2005). Since their isolation and structural elucidation in the 1960s, these compounds have been of great interest within the medicinal chemistry and drug discovery communities, particularly from an anticancer drug development perspective (Chen et al., 2005). Some reports attributed in vitro and in vivo anticancer activities to the cucurbitacins (Fang et al., 1984; Duncan et al., 1996; Chan et al., 2010a; Sun et al., 2010). Jayaprakasam and co-workers demonstrated the cytotoxic properties of cucurbitacins B, D, E and I, isolated from the fruits of Cucurbita andreana, against colon, breast, lung and central nervous system cancer cell lines (Jayaprakasam et al., 2003). Among this class of compounds, cucurbitacin B is the most studied, and while several studies demonstrated its efficacy in different models of cancer, including in vivo tumor xenografts (Liu et al., 2008; Wakimoto et al., 2008; Zhang et al., 2009; Chan et al., 2010), there is disagreement regarding the mechanisms underlying its anticancer activity. Suppression of the oncogene STAT 3 (signal transducer and activator of transcription 3) appears to be related to tumor inhibition (Zhang et al., 2004; Chan et al., 2010b), which does not exclude alternative mechanisms (Yasuda et al., 2010).

Our previous work with Cayaponia racemosa Cong. (Cucurbitaceae), a climber plant found in northeastern Brazil and popularly known as “guardião,” described the isolation of three cucurbitacins, 2,3,16,20(R),25-pentahydroxy-11,22-dioxocucurbita-5-en, 2,3,16,20(R),25-pentahydroxy-22-oxocucurbita-5-en and 2,3,16,20(R),25-pentahydroxy-22-oxocucurbita-5,23(E)-diene, which induced lethality to brine shrimp, inhibited the development of sea urchin eggs and slowed tumor cell proliferation, highlighting their anticancer potential (Dantas et al., 2006). The purpose of this work was to investigate the anticancer activity of 2,3,16,20(R),25-pentahydroxy-22-oxocucurbita-5-en (compound 1) using in vitro and in vivo models.

Materials and methods

Plant material

The plant material was collected in Palmácia, Ceará, Brazil and it was identified by Dr. Edson Paula Nunes. A voucher specimen (30532) was deposited at the Herbário Prisco Bezerra, Federal University of Ceará, Brazil. The air-dried, powdered fruits (0.91 kg) of C. racemosa were extracted exhaustively with EtOH (3 L) at room temperature. After removal of the solvent under reduced pressure, the dark green residue (27.8 g) was fractionated by silica gel (Kieseegel 60 H) column chromatography using hexane, CHCl₃, EtOAc and MeOH as eluents. The EtOAc fraction (8.34 g) contained a mixture of cucurbitacins. A portion (700.0 mg) of this fraction was subjected to reverse-phase hplc on a Hibar RP-18 column (250 × 4 mm) and eluted with MeOH/H₂O (6:4) to yield 2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en (1) (325.0 mg), −687.6° (c 0.43, C₅H₅N), m.p. 141–144°C. Structural elucidation of 1 (Figure 1) was based mainly on spectroscopic analyses using infrared (IR) and ¹H and ¹³C NMR, including special pulse sequences (one- and two-dimensional NMR, such as DEPT 135°, ¹H ¹H COSY, ¹H ¹³C HMQC, ¹H ¹³C HMBC and ¹H ¹H NOESY), and the spectrographs were compared with published data (Stuppner et al., 1981; Abd El-Fattah, 1994).

Figure 1.  A – Chemical structure of 2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en (1) isolated from Cayaponia racemosa. B – Activity of compound 1 and at 2.5 and 5.0 µg/mL on HL60 cells assessed by Trypan blue exclusion. C – Effect of cucurbitacin on the pattern of HL60 cell death, determined by acridine orange and ethidium bromide staining (AO/BE). Negative controls (C) were treated with solvent (DMSO); positive controls (+) were treated with doxorubicin (0.3 µg/mL). The cells were treated for 24 h. *p < 0.05 compared with controls by ANOVA followed by Student Newman–Keuls test.

Antiproliferative activity with human leukemia (HL-60) and peripheral blood mononuclear cells (PBMC)

To investigate the selectivity of compound 1 for normal proliferating cells, the Alamar blue™ assay was performed with human peripheral blood mononuclear cells (PBMC) after 72 h of drug exposure. All studies were performed in accordance with Brazilian research guidelines (Law 196/96, National Council of Health). Heparinized blood (from healthy, non-smoker donors who had not taken any drug for at least 15 days prior to sampling, aged between 18 and 30 year olds) was collected, and PBMC were isolated by the standard method of density-gradient centrifugation over Ficoll-Histopaque. The cells were removed, washed with phosphate buffer saline (PBS) and resuspended in RPMI 1640 medium supplemented with 20% of fetal bovine serum, 100 U/mL penicillin and 100 µg/mL of streptomycin to a final concentration of 3 × 10⁵ cells/mL. Phytohemagglutinin (4%) was added at the beginning of culture, according to Ferreira et al. (2010) with some modifications. After 24 h, the compounds (0.39–25 µg/mL) dissolved in DMSO were added to each well and incubated for 72 h. Doxorubicin (0.009–5 µg/mL) was used as positive control. Control groups received the same amount of DMSO. Twenty-four hours before the end of the incubation, 10 µL of Alamar blue stock solution (Resazurin, Sigma Aldrich Co., 0.312 mg/mL) was added to each well. The absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter^®), and the drug effect was quantified as the percentage of the control absorbances at 570 and 595 nm. HL60 cell line (promyelocytic leukemia) was obtained from the National Cancer Institute (Bethesda, MD) and was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penincillin and 100 µg/mL streptomycin, at 37°C with 5% CO₂. The experiments were performed to investigate the mechanism underlying the cytotoxic effect of a 24 h exposure to cucurbitacin 1 on HL-60. The compound, dissolved in sterilized DMSO, was added to HL-60 cells (3 × 10⁵ cells/mL) to final concentrations of 2.5 and 5.0 µg/mL. Doxorubicin (0.3 µg/mL) was used as a positive control. Cell viability was determined by the Trypan blue dye exclusion test. After incubation of HL-60 cells with cucurbitacin, aliquots were removed from the cultures and stained with Trypan blue. The cells were then scored for dye exclusion in a Neubauer chamber.

Morphological analysis with fluorescence microscopy

Acridine orange/ethidium bromide (AO/EB) staining of HL-60 cells was performed to evaluate the cell death pattern induced by cucurbitacin treatment. After the incubation, the cells were pelleted, and each sample was mixed with 1 µL of aqueous AO/EB solution (100 µg/mL of AO in PBS; 100 µg/mL EB in PBS). Three hundred cells were counted per sample using a fluorescence microscope (Olympus, Tokyo, Japan) and scored as viable, apoptotic or necrotic (Militão et al., 2006).

Analysis of morphological changes by hematoxylin-eosin staining

Untreated or cucurbitacin-treated HL-60 cells were examined for morphological changes by light microscopy (Metrimpex Hungary/PZO-Labimex Modelo Studar lab^®). To evaluate morphology, cells were harvested, transferred to cytospin slides, fixed with methanol for 1 min and stained with hematoxylin-eosin (H/E) (Cury-Boaventura et al., 2004).

In vivo antitumor evaluation

Adult Swiss mice were obtained from the animal facilities of Universidade Federal do Ceará. They were kept in well-ventilated cages (ALESCO, São Paulo) under standard conditions of light (12 h with alternative day and night cycles) and temperature (25 ± 1°C) and were housed with access to commercial rodent stock diet (Purina, São Paulo, Brazil) and water ad libitum. The investigational protocol was approved by the local Ethical Committee on Animal Research at Universidade Federal do Ceará (Process No. 102/2007) and is in accordance with the international standard on the care and use of experimental animals (EEC Directive of 1986, 86/609/EEC). Sixty healthy male mice weighing 23–26 g were subcutaneously implanted with 9-day-old Sarcoma 180 ascites tumor cells (2 × 10⁶ cells/0.5 mL) into the left hind groin. The next day, the mice were randomly divided into six groups (n = 10 per group), and compound 1 dissolved in DMSO 4% was intraperitoneally administered for 7 days at 10 and 25 mg/kg/day doses with or without 5-fluorouracil (5-FU + compound 1, 10 + 10 mg/kg/day, respectively). Negative and positive controls received DMSO 4% and 5-FU (10 mg/kg/day), respectively. On day 8, mice were sacrificed by cervical dislocation, and their organs (kidneys, spleens and livers) and tumors were dissected out, grossly examined for size, color changes and hemorrhage, weighed and preserved in a 10% formaldehyde solution. The inhibition of tumor growth (%) was calculated using the following formula: percent inhibition (%) = [(A–B)/A] × 100, where A is the average tumor weight of the negative control, and B is the average tumor weight of the treated group. To examine morphological changes by light microscopy (Olympus, Tokyo, Japan), small pieces of organs and tumors were processed and embedded in paraffin, and 3–5 µm sections were prepared and stained with hematoxylin-eosin.

Statistical analysis

For cytotoxicity assays, the IC₅₀ values and their 95% confidence intervals were obtained by nonlinear regression using the GraphPad Prism program (GraphPad Software, San Diego, CA). To determine differences, data were compared by one-way analysis of variance (ANOVA) followed by Newman–Keuls test (p < 0.01).

Results

Antiproliferative activity on HL-60 leukemia cell and peripheral blood mononuclear cells (PBMC)

Trypan blue exclusion analysis confirmed the anti-proliferative activity of cucurbitacin 1 (Figure 1A) on HL60. The compound reduced the number of viable cells at both tested concentrations. After a 24-h incubation at 2.5 µg/mL, compound 1 inhibited growth by 40%, without altering the number of non-viable cells (Figure 1A). Only doxorubicin, the positive control, caused cytotoxicity to peripheral blood mononuclear cells with an IC₅₀ of 0.02 µg/mL (0.03 µM), suggesting that the cucurbitacins 1 isolated from C. racemosa was selective for cancer cells, with an IC₅₀ > 25 µg/mL.

Analysis of morphological changes

To further determine whether the growth-inhibitory effect of cucurbitacins was related to the induction of apoptosis and/or necrosis, treated cells were analyzed using AO/EB staining, and the numbers of viable, apoptotic and necrotic cells were determined by fluorescence microscopy. After 24 h of treatment with tested compound, a decreased number of viable cells and an increased number of apoptotic cells were observed in a dose-dependent manner (Figure 1B). The number of viable cells reduced from 85% (control) to 10% after treatment with 5 µg/mL of compound 1. The number of apoptotic cells increased from 20% (control) to 85 and 95% after treatment with 2.5 and 5 µg/mL of compound 1, respectively. Regarding necrosis, no increase was observed after treatment with 5 µg/mL of cucurbitacin (Figure 1B).

Analysis of HL-60 by hematoxilin/eosin staining revealed several drug-induced morphological changes. After 24 h in culture, control cells exhibited a typical non-adherent and vacuolated round morphology. HL60 cells treated with compound 1 showed DNA fragmentation, membrane blebs and eosinophilic granules in the cytoplasm. Doxorubicin (0.3 µg/mL) also reduced cell volume, in addition to causing fragmentation of the DNA and blebbing of the plasma membrane (Figure 2).

Figure 2.  Cucurbitacin 1 induced morphological changes in HL-60 leukemia cells. A – Control cells; B – Cells treated with doxorubicin at 0.3 µg/mL; C and D – Cells treated with compound 1 at 2.5 and 5.0 µg/mL. Black arrows – nuclear fragments showing a periphery of compacted chromatin.

In vivo antitumor evaluation

The effects of compound 1 on tumor growth are presented in Figure 3. A significant reduction in tumor weight (p < 0.01) was found at 25 mg/kg/day for compound 1- and 5-FU + compound 1-treated animals (1.01 ± 0.1 and 0.8 ± 0.1 g, respectively) in comparison to negative controls (1.7 ± 0.1 g), representing 52 and 62% tumor growth inhibition, respectively. In contrast, at 10 mg/kg/day, neither 5-FU nor compound 1 was able to inhibit tumor growth under identical experimental conditions (p > 0.05).

Figure 3.  Tumor mass of mice transplanted with Sarcoma 180 cells after 7 days of treatment by intraperitoneal route with compound 1 (2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en) isolated from Cayaponia racemosa and in association with 5-fluorouracil (5-FU, positive control). Negative control (C) received only DMSO 4%. Data are expressed as mean ± SEM (n = 10 animals/group). Within the square is demonstrated the tumor inhibition capacity expressed as perceptual in comparison with the negative control. *p < 0.01 compared to control by ANOVA followed by Newman–Keuls test.

Examination of tumor morphology from the control group revealed large, polygonal cells with pleomorphic shapes, hyperchromatic nuclei, acidophilic macronucleoli, atypical mitosis and muscle invasion, typical findings in a neoplasm (Figure 4A). Similar results were observed after treatment with 5-FU (Figure 4B) and compound 1 (Figure 4D) at 10 mg/kg/day. Meanwhile, tumors excised from mice treated with 5-FU at 25 mg/kg/day (Figure 4C), compound 1 at 25 mg/kg/day (Figure 4E) and 5-FU + compound 1 at 10 + 10 mg/kg/day (Figure 4F) exhibited extensive areas of coagulative necrosis interspersed with pleormorphic cells. Compound 1, either alone or in association with 5-FU, reduced tumor growth to a similar extent as did the positive control, 5-FU, confirming the in vivo antiproliferative activity of the cucurbitacin.

Figure 4.  Histopathology of tumors excised from Sarcoma 180 transplanted mice after 7 days of intraperitoneal treatment with compound 1 (2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en) isolated from Cayaponia racemosa. A – Negative control (DMSO 4%), B and C – 5-Fluorouracil 10 and 25 mg/kg/day, D and E – Compound 1, 10 and 25 mg/kg/day, F – 5-Fluorouracil + Compound 1 (10 + 10 mg/kg/day), respectively. Magnification, 400×.

In mice, intraperitoneal treatment with compound 1 did not interfere with final body weight and relative liver and kidney weights of the experimental groups (Table 1) when compared to the negative control group (p > 0.05). Moreover, neither mortality nor morbidity was detected during the experiment. 5-FU (25 mg/kg) and the co-treatment with 5-FU caused a reduction in body weight (p < 0.01), and all groups administered compound 1 displayed an increase in relative spleen weight (0.70 ± 0.04 and 0.84 ± 0.03 g, 10 and 25 mg/kg/day, respectively), including the group administered the associated treatment (0.64 ± 0.03 g) (p < 0.01) (Figure 5).

Table 1.  Body effects of compound 1 (2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en) isolated from Cayaponia racemosa on mice transplanted with Sarcoma 180 cells.

Treatment	Dose (mg/kg/day)	Animal weight (g)	Liver	Kidneys	Spleen
			g/100 g body weight
Control	-	34.7 ± 0.7	5.29 ± 0.11	1.39 ± 0.04	0.54 ± 0.03
5-FU	10	33.1 ± 0.7	5.29 ± 0.17	1.41 ± 0.05	0.49 ± 0.03
	25	24.6 ± 0.4*	4.86 ± 0.27	1.28 ± 0.03	0.22 ± 0.01*
Compound 1	10	32.3 ± 1.2	5.56 ± 0.16	1.44 ± 0.06	0.70 ± 0.04*
	25	33.0 ± 1.1	5.41 ± 0.10	1.58 ± 0.04	0.84 ± 0.03*
Compound 1 + 5-FU	10 + 10	30.0 ± 0.9*,**	5.64 ± 0.24	1.54 ± 0.04	0.64 ± 0.03*,**

Data are means ± SEM, n = 10 animals/group, treated for 7 days by intraperitoneal route. Negative control received DMSO 4%.

*p < 0.01 compared to control by ANOVA followed by Newman–Keuls test.

**p < 0.01 compared to 5-FU by ANOVA followed by Newman–Keuls test.

Figure 5.  Histopathology of spleens excised from Sarcoma 180 transplanted mice after 7 days of intraperitoneal treatment with compound 1 (2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en) isolated from Cayaponia racemosa. A – Negative control (DMSO 4%), B and C – 5-Fluorouracil 10 and 25 mg/kg/day, D and E – Compound 1, 10 and 25 mg/kg/day, F – 5-Fluorouracil + Compound 1 (10 + 10 mg/kg/day), respectively. Magnification, 400×.

Hepatic analysis of compound 1-treated mice (10 and 25 mg/kg/day) revealed histological findings similar to that of the negative control group. Comparable alterations were found in the liver of the 5-FU co-treated group, but it also showed sinusoidal hemorrhage and focal infiltration of inflammatory cells, the latter more evident in the groups treated with 5-FU (10 mg/kg/day), which presented focal microvesicular steatosis and enucleated hepatocytes (data not shown). Regarding the kidneys, glomerular and tubular hemorrhage and hyaline cylinders with preservation of the basic outline of glomerular and tubular architecture was observed in all groups. Focal hydropic changes of the tubular epithelium in those mice that received compound 1 at 25 mg/kg/day were also detected.

There was considerable hyperplasia of the splenic white pulp [5-FU + compound 1 (10 + 10 mg/kg/day), Figure 5F] and megakaryocytes (10 mg/kg/day, Figure 5D) in all groups treated with compound 1. Pigments of hemosiderin were observed more frequently after treatment with 25 mg/kg/day of compound 1 (Figure 5E) and in the 5-FU co-treated group (Figure 5F). Meanwhile, negative controls demonstrated no alterations, and 5-FU groups exhibited circumscribed lymphoid follicles, which were more pronounced in the 5-FU co-treated group (Figure 5F).

Discussion

The search for antineoplastic compounds has demonstrated the great pharmacological relevance of plant secondary metabolites (Cragg & Newman, 2005). Curcubitacins possess remarkable pharmacological effects, such as inhibition of cell adhesion (Musza et al., 1994) and T-lymphocyte proliferation (Smit et al., 2000) and anti-inflammatory (Jayaprakasam et al., 2003), immunomodulatory (Attard et al., 2005) and anti-angiogenic activities (Duncan et al., 1996).

Previous work indicated that compound 1 has cytotoxic effects against HL-60 (human leukemia), CEM (human leukemia), HCT-8 (human colon cancer), MCF-7 (human breast cancer) and B-16 (murine melanoma) tumor cell lines. Nevertheless, this compound did not induce hemolysis or alter the development of sea urchin eggs (Dantas et al., 2006).

In the first part of this study, we demonstrated that the cucurbitacin 1 was not cytotoxic to PBMCs at the tested concentrations. In terms of chemotherapy side effects, it is very important to verify whether the drug shows a harmful effect against normal dividing cells, such as proliferating lymphocytes. Additional experiments were performed with HL-60 cells to access the mechanisms underlying the cytotoxic activity of this cucurbitacin on tumor cells. The results of our experiments suggest that the treated cells died via the apoptotic pathway, which is characterized by dissipation of the mitochondrial transmembrane potential and peripheral nuclear condensation, followed by DNA fragmentation and membrane blebbing. Apoptosis is regarded as the preferred process for eliminating undesired cells, and induction of that process is an important strategy in cancer therapies (Leistl & Jäättelä, 2001).

The capacity of the cucurbitacins to induce apoptosis in cultured cells was addressed in several studies (Shan et al., 2010; Yasuda et al., 2010). However, the precise effects and the mechanisms involved in the cellular effects seemed to be greatly affected by the structures of the cucurbitacins and by the cell line studied (Shan et al., 2010; Yasuda et al., 2010). Cucurbitacin B, as previously mentioned, was extensively studied in different models. Doskotch and colleagues were the first to describe the antiproliferative effects of cucurbitacin B using in vitro models (Doskotch et al., 1969). Recently, Chan et al. (2010a) demonstrated the in vitro and in vivo effects of cucurbitacin B against hepatocellular carcinoma. They showed that cucurbitacin B also led to a cyclin D1/cdc2-dependent S-phase arrest followed by apoptosis, with an inhibition of c-Raf activation and unaltered STAT3 phosphorylation. However, the same research group described that the effects of cucurbitacin B on K562 leukemia cells were related to the inhibition of STAT-3 expression (Chan et al., 2010b). Another recent work on the effects of cucurbitacin E on pancreatic cells demonstrated an association with inhibition of STAT 3 (Sun et al., 2010). The STAT proteins have dual roles as cytoplasmic signaling proteins and nuclear transcription factors. Persistent activation of Stats, particularly STAT3, has been implicated in the pathogenesis of a whole spectrum of malignancies, including T-cell lymphoma (Blaskovich et al., 2003). Previous studies showed that cucurbitacin I decreased the expression of Stat3 in Sz cells (T-cell lymphoma) and ALK⁺ALCL cells (ALK-positive anaplastic large cell lymphoma). Furthermore, cucurbitacin I inhibited the growth of the aforementioned cells by inducing apoptosis (Shi et al., 2006). These data reinforce the anticancer potential of cucurbitacins, while also highlighting the diverse mechanisms that can be triggered by this class of compounds.

Herein, we also reported, for the first time, the antitumor activity of compound 1, obtained from C. racemosa, in mice bearing the Sarcoma 180 tumor, a mouse-derived tumor widely exploited in antitumor research in vivo (Sato et al., 2005; Magalhães et al., 2010). Compound 1, either alone or in association, with 5-FU, reduced tumor growth to a similar extent as did the positive control, 5-FU, confirming the in vivo antiproliferative activity of the cucurbitacin. Cucurbitacin B, another molecule isolated from C. racemosa, dose-dependently inhibited hepatocellular carcinoma cell (BEL-7402) tumor growth in vivo at 0.1, 0.5 and 1 mg/kg by mouth (Chan et al., 2010a). In association with docetaxel, cucurbitacin B also d antitumor effects in the Hep-2 (laryngeal cancer) xenograft model (Liu et al., 2008). Because such combined biological therapies hold promise for controlling tumor proliferation and drug-resistance, the development of combination chemotherapies is expected to increase the response rate and the frequency of long-term survival (Bezerra et al., 2008).

The kidney and liver have been proposed as key organs for the metabolism of environmental toxic substances (Scheuer & Lefkowitch, 2000). Furthermore, these organs also possess a pronounced regenerative capacity: even when necrosis is found with conjunctive tissue preservation, there is often complete tissue restoration (Kumar et al., 2004). In this context, the activity of compound 1 in vivo was related to moderate effects on the liver and kidney, and all observed morphological alterations in cucurbitacin-treated animals could be considered potentially reversible. Additionally, compound 1 presented effects on the spleen of treated animals, leading to hyperplasia of the splenic white pulp and an increased number of megakaryocytes, which is suggestive of an immunostimulatory effect that may also contribute to its in vivo antitumor properties (Imboden, 1988).

Conclusions

In conclusion, the present data highlight the importance of cucurbitacins as an emerging class of potential anticancer lead molecules, exhibiting an antiproliferative effect on leukemia cells and an antitumor effect in vivo that is associated with reversible toxic effects.

Acknowledgments

We are grateful to Silvana França for technical assistance. The English was edited by American Journal Experts (key # ADC5-52E4-E64C-F4E9-24BD).

Declaration of interest

We wish to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Financiadora de Estudos e Projetos (FINEP), Banco do Nordeste do Brasil (BNB) and Instituto Claude Bernard (InCb) for financial support in the form of grants and fellowship awards. The authors report no declarations of interest.