ANTICANCER ACTIVITY OF β-LAPACHONE AN INGREDIENT OF PAU D’ARCO HERB

ANTICANCER ACTIVITY OF β-LAPACHONE AN INGREDIENT OF PAU D’ARCO HERB

Dr Evangelos Kontargiris,

Bsc, Biomedical Sciences, PhD in cellular and molecular physiology

Pau d’arco in a herb, popular in South America, which is made by the inner bark of a tree named Tabebuia, and contain the active substances called naphthoquinones which are benzoic acid derivatives. It was found by in vitro studies in cancer cells that one naphthoquinone called β–lapachone possesses powerful anticancer properties via specific cytotoxic mechanisms of molecular biology and biochemical pathways.

Pau d’arco in a well-known herb which is available in tea bags, in capsules and powder from health products stores. It is also known as lapacho, ipe roxo and taheebo. The herb is made by the inner bark of a large tree of various types named Tabebuia, with flowers of pink, purple and yellow colors which could be found in Brazil and Argentina. In South America pau d’arco has been used against cancer, syphilis, gonorrhea, malaria, flu, infections, inflammation and at least one type of lupus. It has been also used against bacteria, fungi and also as a diuretic and as an astringent.^{1, 2}

It was discovered that various types of active substances are contained in pau d’arco which are called naphthoquinones, are benzoic acid derivatives and act synergistically in cancer therapy^1,2

One recent study showed that treatment of breast cancer cells with the aqueous extract of taheebo from the inner bark of the Tabebuia avellandae tree³ (Figure 1) resulted in a dose/time-dependent growth inhibition by S phase arrest (where DNA replication takes place) and initiation of apoptosis by chromatin condensation (Figure 2) in these cells.⁴

 

Image 1.  Flowers of Tabebuia avellanedae tree3

Image 2. Transmission electron microscopy based cellular morphology of apoptotic and necrotic cells. (A) Untreated control, (B) treated with 1.5 mg/ml of Taheebo for 24 h. Note the presence of peripheral condensation and central fragmentation of nuclear chromatin in the apoptotic cell, and the presence of membrane blebbing and cytoplamic lysosomes in the necrotic cells. ⁴

According to laboratory in vitro studies which took place in cells of various types of cancer, one naphthoquinone called β-lapachone (Figure 3) which is contained in pau d’arco from Tabebuia avellandae tree³, possesses powerful anticancer properties.^{2, 5} These studies revealed specific mechanisms of molecular biology and possible biochemical pathways via which the cytotoxic action of β-lapachone against cells is done and therefore its anti-cancer activity is explained in depth.

 

Image 3. Chemical structure of β-lapachone.⁵

Firstly, one study was done in the cultured human prostate carcinoma cells PC-3 and according to the results β-lapachone caused inhibition of the proliferation and apoptosis induction of the cells and also arrested the cell cycle progression at the G1 phase (Figure 4). Under normal conditions the cells in G1 phase of the cell cycle are increased in size and are prepared for DNA synthesis. These results provide convincing evidence that b-lapachone exerts its effect on cell cycle progression of PC-3 cells by two pathways: First, by a significant decrease in the phosphorylated forms of tumor suppressor retinoblastoma protein pRB, that lead to its inhibitory effect of the E2F-1 (transcription factor) dependent transcriptional activity of the genes that control the transition from the G1 to S phase and subsequent DNA synthesis. Second, by an increase in the p21 expression that leads to its increased binding with cyclin-dependent kinase, cyclin/Cdk, complexes which results in a marked decrease in their kinase activities. ⁶

 

Image 4. β-lapachone-induced morphological changes in PC-3 cells. The cells were incubated with 3 μM and 6 μM β-lapachone for 6 h. After 24 h incubation in a normal medium, the cells were sampled, stained with Wright dye, and examined under light microscopy. Magnification, x 200. ⁶

Another study which took place in a different cell line of cultured human prostate carcinoma cells called DU145 showed that exposure of the cells to β-lapachone resulted in growth inhibition and induction of apoptosis in a dose-dependent manner (Figure 5). The increase in apoptosis was associated with a dose-dependent up-regulation in pro-apoptotic Bax expression, and down-regulation of anti-apoptotic Bcl-2. Moreover it was found that β-lapachone decreased the levels of cyclooxygenase (COX)-2 mRNA and protein expression which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. Prostaglandins are known to play an important role in many functions of the body including inflammation. Furthermore, β-lapachone treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Telomarase is an enzyme that conserves and repairs the lengths of telomeres which are the final ends of chromosomes. This enzyme adds repeating small fragments of DNA in the final ends of chromosomes and therefore the genetic material is not destroyed or got lost during the cellular division.^{7, 8}

 

Image 5. Morphological changes by β-lapachone treatment in human prostate carcinoma DU145 cells. Cells were incubated with variable concentrations of β-lapachone. After 48 h incubation, cells were sampled and examined under light microscopy. Magnification, ×200.⁷

Concerning other types of cancer it was found that exposure of the human colon carcinoma tumor cell line HCT-116 to β-lapachone resulted in growth inhibition and induction of apoptosis in a dose-dependent manner (Figure 6). This increase in apoptosis was associated with a decrease in Bcl-2 protein expression, and the marked decrease of nuclear protein levels of nuclear factor κB (NF-κB), a protein complex which controls many biological activities in cells including apoptosis.⁹ Moreover, exposure of the human lung carcinoma cell line A549 to β-lapachone resulted in growth inhibition and induction of apoptosis in a time- and dose-dependent manner. This increase in apoptosis was associated with a decrease and a downregulation in Bcl-2, in the levels of human telomerase RNA (hTR) and in c-myc expression and also in an increase of Bax.¹⁰ Furthermore it is known that β-lapachone inhibits the viability of the human hepatoma cell line HepG2 by inducing apoptosis by down-regulation of anti-apoptotic Bcl-2 and Bcl-XL and up-regulation of pro-apoptotic Bax expression (Figure 7). In the same study it is revealed that these apoptotic mechanisms were mediated by a modulation of the Bcl-2 family and an activation of caspase-3 and -9 without any effects from the inhibitor of apoptosis proteins (IAPs) family and Fas/FasL system, suggesting that β-lapachone-induced apoptosis is mediated, at least in part, by the mitochondrial signaling pathway. ^{11 In another study it was found that} β-lapachone inhibited the viability of human bladder carcinoma T24 cell line cells by inducing apoptosis which is mediated, at least in part, by the mitochondrial-signaling pathway. That’s because β-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression was also associated with activation of caspase-3 and caspase-9, and the inhibition of Fas and FasL levels in these cells.¹² Finally another article revealed that β-lapachone treatment induces direct cytotoxicity in human leukemia cells (U937, K562, HL60, and THP-1) through activation of caspase-3. The observed induction of cell death was associated with decreased telomerase activity, which was ascribed to down-regulation of telomerase reverse transcriptase. ¹³

 

Image 6. Morphological changes of human colon cancer HCT-116 cells after treatment with β-lapachone. Cells were incubated with variable concentrations of β-lapachone (1-5 μΜ for 24 hours). After 24 h incubation cells were sampled and examined under light microscopy. Magnification x200. ⁹

Image 7. β-Lapachone induces apoptosis in HepG2 cells. Cells were incubated with variable concentrations of β-lapachone. After a 48-hour incubation cells were sampled, fixed, and stained with DAPI. After a10-minute incubation at room temperature, stained nuclei were observed under a fluorescent microscope using a blue filter. Magnification x 400. The untreated control cells displayed intact nuclear structures, while cells treated with β-lapachone had chromosomal condensation and the formation of apoptotic bodies in a concentration-dependent manner.¹¹

Apart from its cytotoxic activity in various types of cancer β-lapachone was found to have anti-metastatic and anti-invasive abilities. According to the results of a study which was done in human hepatocarcinoma cell lines HepG2 and Hep3B, β-lapachone dramatically increased the expression of early growth response gene-1 (Egr-1), of throbospondin-1 (TSP-1) and of E–cadherin. The tumour suppressor gene Egr-1 regulates the expression of a diverse array of genes involved in tumor metastasis including TSP-1 which in turn is a potent inhibitor of neovascularization that limits tumor growth. Neovascularization is the development of capillaries in tissues which in normal conditions are not present and it happens in tumor tissues. E-cadherin is a cell-cell adhesion molecule that is especially expressed on the membranes of epithelial cells and decreased expression of it has been reported in invasion and metastasis of cancers. ¹⁴

References

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