For citation purposes: Hsieh YJ, Leu YL, Chang CJ. The anti-cancer activity of Kalanchoe tubiflora. OA Alternative Medicine 2013 Aug 01;1(2):18.

Critical review

 
Herbalism

The anti-cancer activity of Kalanchoe tubiflora

YJ Hsieh1*, YL Leu2, CJ Chang3*
 

Authors affiliations

(1) Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Taiwan

(2) Graduate Institute of Natural Products, Chang Gung University, Taiwan

(3) Department of Molecular Biology and Human Genetics, Tzu Chi University, Taiwan

*Corresponding author Email: hij@mail.tcu.edu.tw; cjchang@mail.tcu.edu.tw

Abstract

Introduction

Uncontrolled cell proliferation is a common feature of human cancer. Most of the drugs used currently in therapeutics strategies are classified as antiproliferative drugs via apoptosis pathway. Mitotic catastrophe is a distinct non-apoptotic mechanism often triggered in cancer cells and tissues in response to anti-cancer drugs. As of date, little research exists to show Herbal extracts or plant-derived medicines are an important source of effective anti-cancer agents, particularly for treating mitotic catastrophe. This article explores how mitotic catastrophe is triggered by Kalanchoe tubiflora and opens avenues for further research by presenting comparable results obtained so far.

Conclusion

For the past five years, we have been investigating the anti-cancer effects of Kalanchoe tubiflora, particularly to understand its action mechanism, and have analysed its efficacy through various in vitro and in vivo tests. Our preliminary studies show that Kalanchoe tubiflora is a potential anti-cancer agent and merits further investigation. The extract of Kalanchoe tubiflora inhibits cell proliferation and reduces cell viability through two mechanisms. First, it disrupts centrosome integrity and induces multipolarity; second, it perturbs chromosome alignment at metaphase. Both mechanisms specifically target mitotic cells, which leads to cell death.

Introduction

Research on medicinal plants has shown that they are an important source of effective anti-cancer agents. More than half of the drugs used in clinical trials for anti-cancer activity are derived from natural sources[1]. Kalanchoe is a genus of the Family Crassulaceae. Various species of Kalanchoe are used medicinally in Southeast Asia, specifically Indo-China and Philippines. Plants of this genus are mentioned in folklore and are used as traditional medicines for treating fever, abscesses, bruises, contused wounds, coughs, skin diseases, infections, hypertension, rheumatism and inflammation[2,3] and, in particular, tribes in the state of Kerala, India, use these plants for treating cancer symptoms. A variety of bufadienolide compounds have been isolated from various Kalanchoe species, which showed strong antitumour-promoting activity[4,5,6].

In one of the above cases, Kalanchoe hybrida was used. It is the hybrid of Kalanchoe daigremontiana and Kalanchoe tubiflora (KT) and is naturalized throughout the island of Taiwan. On the basis of the aforementioned studies, KT, one of the origin sources of the hybrid, was selected as a target given its biological activities. KT is used as a wound-healing agent in traditional South Brazilian medicine[7]. Few cardenolide and bufadienolide glycoside compounds were isolated from KT in Kuo group. Four of the isolated compounds have the capacity to arrest HL-60 cells in G2/M phase[8]. In our study, we used KT as an example to discuss agents that target mitotic cells.

Discussion

The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.

Anti-cancer activity

Schmidt et al. carried out a study to establish the biological properties of Brazilian plants used in wound healing[7]. By studying the different bioassays identified, Schmidt et al. found that KT did not show much promise as a potential treatment agent. In fact, KT showed a toxic effect in the MTT assay. This is consistent with the findings of our study[9]. Toxicity of KT was tested in different cell lines and different dosages. The cell viability for all cell lines was found to be 20% below the concentration level of 20μg/ml. Hence, there is sufficient evidence to suggest that KT extract effectively inhibits growth of different cancer cell lines.

The scratch assay in Schmidt’s study showed that the n-hexanic extract of KT had no activity in the scratch assay at a concentration of 10μg/ml. The ethanoic extract showed a moderate ‘wounding-healing’ effect. However, in our study we found that KT extract completely blocked ‘wound healing’ at a concentration of 50μg/ml. Note that the scratch assay does not distinguish between migration and proliferation. However, in terms of anti-cancer activity, KT is a promising agent for anti-proliferation and/or anti-migration.

Anti-cancer mechanism

Mitosis is a highly coordinated process in which two copies of one chromosome are moved away from each other to the opposite poles of cells. Sister-chromatid segregation depends on a complex molecular scaffold of the mitotic spindle. Disruption of the mitotic spindle structure precludes proper alignment of chromosomes and activates the spindle assembly checkpoint and mitotic arrest, which in turn provides time for cells to correct the attachment error between the microtubule and kinetochore. However, prolonged mitotic delay is often followed by cell death in mitosis. This is the strategy to kill cancer cells by perturbing mitotic spindle assembly. Microtubules play an important role in cell proliferation, trafficking and migration. Microtubules are a basic component of mitotic spindle. This is why agents that bind to microtubules are used in cancer therapy. Two clinical anti-cancer drugs, taxanes and vinca alkaloids, are natural products that affect microtubule dynamics and cause abnormal spindle formation[10,11,12]. However, microtubules are also present in interphase cells. Microtubule binding agents also perturb the micro-tubule network of interphase cells and cause neurotoxic effects[13].

The microtubule binding agents have side-effects unrelated to antimitotic effects. This warrants searching for more mitosis-selective drugs. The mitotic-selective approach targets proteins that function only in mitosis. Some of the drugs that target the six mitotic proteins are currently undergoing clinical trials. These mitotic proteins include two motor proteins (Eg5, CENP-E) and four kinases (Aurora A, Aurora B, Cdk1, Plk1)[14,15]. Eg5 is a plus end-directed motor protein that drives centrosome separation in prophase[16]. Inhibition of Eg5 results in monopolar spindles causing mitotic arrest in a spindle-assembly-checkpoint-dependent manner[17,18]. CENP-E is a kinetochore-associated protein that stabilizes the interactions between microtubules and kinetochore during mitosis[19]. It functions exclusively in mitosis. Inhibition of CENP-E leads to misaligned chromosomes in meta-phase[20]. CDK1 is the main regulator for mitotic entry in mammalian cells[21]. Plk1, Aurora A and Aurora B regulate mitotic entry and ensure that chromosome segregation and cytokinesis events take place[22].

In our previous study, we found that KT extract also has the capacity to arrest cells in the G2/M phase. Drugs that bind to microtubules are able to arrest cells in G2/M. However, KT extract does not disturb the microtubule organization in interphase or spindle formation in mitosis. Instead, KT extract treatment resulted in multipolar spindles (Figure 1A) and chromosome misalignment (Figure 1B). These defects activate mitotic checkpoints and cause mitotic arrest[23]. The arrested mitotic cells are often followed by cell death in mitosis. Some of the mitotic defective cells could exit mitosis and end up in a tetraploid or aneuploid state. The fate of those mitotic checkpoint slippage cells could be apoptosis, senescence or necrosis[24]. Various compounds isolated from KT have been summarized in this critical review (Table 1)[8,25].

Mitotic cells. (A) A cell with multipolar spindle. (B) The chromosomes of a metaphase cell. Arrow points to the misalignment of chromosomes.

Table 1

Phytochemicals isolated from Kalanchoe tubiflora

Conclusion

Finding ways to develop traditional herbal medicine is a matter of great urgency. Plants have chemical defence mechanisms that synthesize a wide variety of compounds that can be used to perform important biological functions and to defend against attack from predators. Many of these phytochemicals have beneficial effects, but some can have adverse or lethal effects on humans. However, no single chemical can effectively treat human diseases without side-effects. Isolation, identification and bioactive tests of pure compounds are standard strategies utilized in studying natural products. In many cases, scientists are not sure what specific ingredient in a particular herb can treat diseases. Whole herbs contain many ingredients that likely work together to produce a beneficial effect. Herbalists tend to use extracts from different parts of plants, such as roots or leaves, but do not isolate particular phytochemicals. They often reject the notion of a single active ingredient, arguing that the different phytochemicals present in many herbs will interact to enhance the therapeutic effects of the herbs and dilute toxicity. However, the activities of compounds isolated from plants are not necessarily parallel to the phenotypes observed in the traditional use of these plants.

A major aspect of traditional Chinese medicine focuses on restoring the homeostasis of the body to maintain health rather than treating a particular disease or medical condition. If used correctly, herbs can cure a variety of conditions, and in some cases, may have fewer side-effects than some of the conventional medications. Here we would like to draw an open question. Shall we keep doing separation? In understanding the role of medicinal herbs in human physiology, one might end up with putting all the isolated components together in order to reveal how therapy works. Hence, we still have a long way to go in determining the most effective modalities of treatment that uses medicinal herbs. More scientific data and evidence-based research are needed to determine the effectiveness of medicinal herbs.

Competing interests

None declared.

Conflict of interests

None declared.

Authors Contribution

All authors contributed to the concept on, design, and preparation of the manuscript, as well as read and approved the final manuscript.

A.M.E

All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

References

  • 1. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012 Mar 23;75(3):311-35.
  • 2. Supratman U, Fujita T, Akiyama K, Hayashi H. Insecticidal compounds from Kalanchoe daigremontiana x tubiflora. Phytochemistry 2001 Sep;58(2):311-4.
  • 3. Lans CA . Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus. J Ethnobiol Ethnomed 2006 Oct 13;245.
  • 4. Lai ZR, Ho YL, Huang SC, Huang TH, Lai SC, Tsai JC. Antioxidant, anti-inflammatory and antiproliferative activities of Kalanchoe gracilis (L.) DC stem. Am J Chin Med 2011;39(6):1275-90.
  • 5. Costa SS, Jossang A, Bodo B, Souza ML, Moraes VL. Patuletin acetylrhamnosides from Kalanchoe brasiliensis as inhibitors of human lymphocyte proliferative activity. J Nat Prod 1994 Nov;57(11):1503-10.
  • 6. Supratman U, Fujita T, Akiyama K, Hayashi H, Murakami A, Sakai H. Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora. Biosci Biotechnol Biochem 2001 Apr;65(4):947-9.
  • 7. Schmidt C, Fronza M, Goettert M, Geller F, Luik S, Flores EM. Biological studies on Brazilian plants used in wound healing. J Ethnopharmacol 2009 Apr 21;122(3):523-32.
  • 8. Huang HC, Lin MK, Yang HL, Hseu YC, Liaw CC, Tseng YH. Cardenolides and bufadienolide glycosides from Kalanchoe tubiflora and evaluation of cytotoxicity. Planta Med 2013 Sep;79(14):1362-9.
  • 9. Hsieh YJ, Yang MY, Leu YL, Chen C, Wan CF, Chang MY. Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus. BMC Complement Altern Med 2012 Sep 10;12149.
  • 10. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov 2010 Oct;9(10):790-803.
  • 11. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004 Apr;4(4):253-65.
  • 12. Manfredi JJ, Horwitz SB. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol Ther 1984;25(1):83-125.
  • 13. Canta A, Chiorazzi A, Cavaletti G. Tubulin: a target for antineoplastic drugs into the cancer cells but also in the peripheral nervous system. Curr Med Chem 2009;16(11):1315-24.
  • 14. Janssen A, Medema RH. Mitosis as an anti-cancer target. Oncogene 2011 Jun 23;30(25):2799-809.
  • 15. Manchado E, Guillamot M, Malumbres M. Killing cells by targeting mitosis. Cell Death Differ 2012 Mar;19(3):369-77.
  • 16. Sawin KE, LeGuellec K, Philippe M, Mitchison TJ. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 1992 Oct 8;359(6395):540-3.
  • 17. Blangy A, Lane HA, d’Herin P, Harper M, Kress M, Nigg EA. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 1995 Dec 29;83(7):1159-69.
  • 18. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999 Oct 29;286(5441):971-4.
  • 19. McEwen BF, Chan GK, Zubrowski B, Savoian MS, Sauer MT, Yen TJ. CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells. Mol Biol Cell 2001 Sep;12(9):2776-89.
  • 20. Putkey FR, Cramer T, Morphew MK, Silk AD, Johnson RS, McIntosh JR. Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Dev Cell 2002 Sep;3(3):351-65.
  • 21. Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci 2005 Nov;30(11):630-41.
  • 22. Taylor S, Peters JM. Polo and Aurora kinases: lessons derived from chemical biology. Curr Opin Cell Biol 2008 Feb;20(1):77-84.
  • 23. Lara-Gonzalez P, Westhorpe FG, Taylor SS. The spindle assembly checkpoint. Curr Biol 2012 Nov 20;22(22):R966-80.
  • 24. Vitale I, Galluzzi L, Castedo M, Kroemer G. Mitotic catastrophe: a mechanism for avoiding genomic instability. Nat Rev Mol Cell Biol 2011 Jun;12(6):385-92.
  • 25. Huang HC, Huang GJ, Liaw CC, Yang CS, Yang CP, Luo CL. A new megastigmane from Kalanchoe tubiflora (Harvey) Hamet. Phytochemistry Letters 2013;6(3):379-82.
Licensee to OAPL (UK) 2013. Creative Commons Attribution License (CC-BY)

Phytochemicals isolated from Kalanchoe tubiflora

I Flavonoids III Steroids
5,7,8,4’-pentahydroxy-3’-methoxyflavone ß-sitosterol-O-D-glucoside
4’-methoxyherbacetin Kalantubolide A
Quercetin Kalantubolide B
II Benzenoids Kalantuboside A
Trans-ferulic acid Kalantuboside B
Cinnamic acid Stigmasterol-O-D-glucoside
Gallic acid Bersaldegenin-1-acetate
4-O-ethylgallic acid Bersaldegenin-1,3,5-orthoacetate
Syringic acid Bryotoxin C
Vanillic acid IV Others
Methyl gallate Taurolipid C
3,4-dimethoxyphenol Methyl (9S,10R,11E,13R)-9,10,13-Trihydroxyocradec-11-enoate
Phloroglucinol
3,4-dihydroxyallylbenzene Methyl (9S,10R,11E,13S,15E)-9,10,13-trihydroxyocradec-11,15-dienoate
(6S,7R,8R,9S)-6-oxaspiro-7,8-dihydroxymegastigman-4-en-3-one(tubiflorone)