In Vitro Comparative Study on Antineoplastic Effects of Pinoresinol and Lariciresinol on Healthy Cells and Breast Cancer-Derived Human Cells.

Background: Herbal medicines are the preferred anticancer agents due to their lower cytotoxic effects on healthy cells. Plant lignans play an important role in treating various diseases, especially cancer. The present study aimed to evaluate the effect of podophyllotoxin, pinoresinol, and lariciresinol on cellular toxicity and inducing apoptosis in fibroblasts, HEK-293, and SkBr3 cell lines. Methods: An in vitro study was conducted from 2017 to 2019 at the Faculty of Biological Sciences, Tarbiat Modares University (Tehran, Iran). The cell lines were treated for 24 and 48 hours with different concentrations of lignans. Cell viability and apoptosis were examined using MTT and flow cytometry, respectively. Expression levels of cell cycle and apoptosis regulator genes were determined using quantitative real-time polymerase chain reaction. Data were analyzed using a two-way analysis of variance followed by Tukey's HSD test. P<0.05 was considered statistically significant. Results: Podophyllotoxin significantly increased apoptosis in fibroblast cells compared to pinoresinol and lariciresinol (P<0.001). The percentage of cell viability of fibroblast cells treated for 48 hours with pinoresinol, lariciresinol, and podophyllotoxin was reduced by 49%, 47%, and 36%, respectively. Treatment with pinoresinol and lariciresinol significantly overexpressed pro-apoptotic genes and underexpressed anti-apoptotic genes in SkBr3 cells (P<0.001). SkBr3 cells treated with lariciresinol significantly reduced gene expression (P<0.001). Conclusion: Pinoresinol and lariciresinol can potentially be used as new therapeutic agents for the treatment of breast cancer.

Physiological, biochemical, and molecular responses of Linum album to digested cell wall of Piriformospora indica.

Plants synthesize a variety of metabolites in response to biotic elicitors. To comprehend how the digested cell wall of Piriformospora indica affects the response of ROS burst, antioxidant enzymes, amino acids profiling, and phenylpropanoid compounds such as lignans, phenolic acids, and flavonoids in Linum album hairy roots; we accomplished a time-course analysis of metabolite production and enzyme activities in response to CDCW and evaluated the metabolic profiles. The results confirms that CDCW accelerates the H2O2 burst and increases SOD and GPX activity in hairy roots. The HPLC analysis of metabolic profiles shows that the H2O2 burst shifts the amino acids, especially Phe and Tyr, fluxes toward a pool of lignans, phenolic acids, and flavonoids through alterations in the behavior of the necessary enzymes of the phenylpropanoid pathway. CDCW changes PAL, CCR, CAD, and PLR gene expression and transiently induces PTOX and 6MPROX as the main-specific products of PAL and PLR genes expression. The production of phenolic acids (e.g., cinnamic, coumaric, caffeic, and salicylic acid) and flavonoids (e.g., catechin, diosmin, kaempferol, luteolin, naringenin, daidzein, and myricetin) show different behaviors in response to CDCW. In conclusion, our observations show that CDCW elicitation can generate H2O2 molecules in L. album hairy roots and consequently changes physiological, biochemical, and molecular responses such as antioxidant system and the specific active compounds such as lignans. Quantification of metabolic contents in response to CDCW suggests enzyme and non-enzyme defense mechanisms play a crucial role in L. album hairy root adaptation to CDCW. A summary revealed that the correlation between H2O2 generation and L. album hairy root defense system under CDCW. Increase of H2O2 generation led plant to response against oxidative conditions. SOD, and GPX modulated H2O2 content, Phe, and Tyr shifted to the phenylpropanoid compounds as a precursor of PAL and TAL enzyme, the predominant phenylpropanoid compounds controlled oxidative conditions, and the other amino acids responsible for amino acid synthesis and development stages.

Time-dependent behavior of phenylpropanoid pathway in response to methyl jasmonate in Scrophularia striata cell cultures.

KEY MESSAGE: MeJA triggers a time-dependent behavior of the phenylpropanoid compounds. Plant cells produce a large number of metabolites in response to environmental factors. The cellular responses to environmental changes are orchestrated by signaling molecules, such as methyl jasmonate (MeJA). To understand how the MeJA changes the behavior of amino acids, carbohydrates, and phenylpropanoid compounds such as phenolic acids, phenylethanoid-glycosides, and flavonoids in Scrophularia striata cells; we monitored the metabolic responses for different times of exposure. In this study, we performed a time course analysis of metabolites and enzymes in S. striata cells exposed to MeJA (100 µM) and evaluated the metabolic flux towards carbon-rich secondary metabolites production. Moreover, we calculated the biosynthetic energy cost for free amino acids. Our results indicated that MeJA accelerates the sucrose degradation and directs the metabolic fluxes towards a pool of flavonoids and phenylethanoid glycosides through a change in enzyme behavior in the entry point and center of the phenylpropanoid pathway. MeJA also decreased and then raised the amino acid biosynthesis cost in S. striata cells in a time-dependent manner, indicating the cells evolve to utilize amino acids more economically by reducing cell growth. Finally, we classified the marked changes in the metabolites level and enzyme activities into three groups including early-, late-, and oscillatory-response groups to MeJA and summarized our findings as a model depicting pathway interactions during MeJA elicitation. Determination of metabolic levels in response to MeJA suggests that the changes in metabolic responses are time-dependent.

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth.

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem's inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150 mg l-1 on the 8th day. The highest content of BA was 1.3 mg g-1 DW after treatment with 1 mg l-1CCC on the 4th and 6th days and 200 mg l-1CTS NF on the 10th day. The highest B content (0.94 mg g-1DW) was obtained in the treated hairy root by 2 mg l-1 CCC after 4 and 6 days.

RNAi-mediated silencing of pinoresinol lariciresinol reductase in Linum album hairy roots alters the phenolic accumulation in response to fungal elicitor.

Lignans are diphenolic compounds produced in plants via coupling of two coniferyl alcohol molecules with the aid of a dirigent protein to form pinoresinol (PINO). The latter is reduced via lariciresinol (LARI) to secoisolariciresinol by the bifunctional pinoresinol-lariciresinol reductase (PLR). In this study, we clarified the consequences of altered lignan biosynthesis on amino acids, phenolics compounds and lignin in the hairy roots of Linum album with an ihpRNAi construct to silence PLR gene expression. Down-regulation of PLR-La1 resulted in up to an 8.3 and 3.3-time increased PINO and LARI content respectively, and reduced levels of podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6-MPTOX). By Suppression of PLR expression, the metabolites belonging to shikimate and phenylpropanoid pathways are conducted to phenolic compounds and lignin accumulations. Although PINO and LARI were induced in response to fungal elicitor, the accumulation of PTOX and 6-MPTOX did not occur in PLR down-regulated roots. Our result also demonstrated variation in amino acids, phenolic compounds and lignin levels in presence of the fungal elicitation in PLR down regulated-roots. This data assert the accumulation of aryltetralin lignans in interactions with plant pathogens by PLR activity and the importance this enzyme for defense against pathogens in L. album.

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors.

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L-1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L-1 of chitosan was found to be 5.9 × (6.5 mg g-1 DW) higher over control cells. Treating the cells with 2 mg L-1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g-1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.

Water stress alleviation by polyamines and phenolic compounds in Scrophularia striata is mediated by NO and H2O2.

Plants respond to water stress through a variety of mechanisms, depending on metabolites preferences and their available resources. This work was performed to elucidate the cross-talk between signaling molecules (polyamines (PAs), hydrogen peroxide (H2O2) and nitric oxide (NO)), phenolic compounds and osmolytes (phenylethanoid glycosides (PhGs), phenolic acids, flavonoids, soluble sugars and amino acids) under water stress in Scrophularia striata plants. The results revealed that PAs, NO levels were enhanced in the plants, earlier in response to polyethylene glycol-induced water stress. The antioxidative mechanisms with increased activity of catalase (CAT), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) and also phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), as key enzymes in phenolic pathway were deployed in response to the stress. Mannose, glucose, xylose/rhamnose which are involved in PhGs biosynthesis as well as in serving osmotic adjustment were modulated. The elevated content of arginine and methionine as PAs precursors and tyrosine and phenylalanine as PhGs precursors was enhanced by water stress and was significantly associated with PAs and PhGs accumulations. Metabolic profiling revealed new information about relationship between stress signal molecules; PAs, NO and H2O2, osmolytes (sugers, PhGs) and phenolic compounds which involved in the improvement of water stress tolerance in S. striata.

Modulation of Pb-induced stress in Prosopis shoots through an interconnected network of signaling molecules, phenolic compounds and amino acids.

Lead (Pb) is a hazardous heavy metal present in the environment which elicits oxidative stress in plants. To characterize the physiological and biochemical basis of Pb tolerance, Prosopis farcta seedlings were exposed to Hoagland's solutions at six different Pb concentrations (0, 80, 160, 320, 400 and 480 µM) for different periods of time. As expected, application of Pb significantly increased hydrogen peroxide (H2O2) content. In response, P. farcta deployed the antioxidative defence mechanisms with significantly higher activities of superoxide dismutase (SOD), enzymes related to H2O2 removal, and also the increases in proline as a solute marker of stress. Increases were observed in nitric oxide (NO) production which could also act in triggering defense functions to detoxify Pb. Enhanced phenylalanine ammonia-lyase (PAL) activity at early days of exposure to Pb was correlated with increases in phenolic compounds. Significant increases in phenolic acids and flavonoids; daidzein, vitexin, ferulic acid and salicylic acid were observed with Pb treatment. Furthermore, the stress effects were followed by changes in free amino acid content and composition. Aspartic acid and glycine content was increased but glutamic acid significantly decreased. It is likely that stress signal transduction by NO and H2O2 mediated defence responses to Pb by coordination of antioxidative system and metabolic pathways of phenylpropanoid and amino acids.

Lignan enhancement in hairy root cultures of Linum album using coniferaldehyde and methylenedioxycinnamic acid.

Feeding experiments with hairy root cultures of Linum album have established that the extracellular coniferaldehyde is a good precursor for production of two lignans: lariciresinol (LARI) and pinoresinol (PINO). The accumulation of the LARI, PINO, and podophyllotoxin (PTOX) in hairy roots were enhanced about 14.8-, 8.7-, and 1.5-fold (107.61, 8.7 and 6.42 µg g(-1) Fresh Wight), respectively, by the addition of coniferaldehyde (2 mM) to the culture media (after 24 hr). This result was correlated with an increase pinoresinol/lariciresinol reductase (PLR) expression gene and cinnamyl alcohol dehydrogenase (CAD) activity in the fed hairy roots. Adding 3,4-(methylendioxy)cinnamic acid (MDCA) precursor did not influence on the lignans accumulation, but the lignin content of the hairy roots was increased. Moreover, the expression genes of phenylalanine ammonialyase (PAL), CAD, and cinnamoyl-CoA reductase (CCR) were influenced after feeding hairy roots with MDCA.

6-Methoxy Podophyllotoxin Induces Apoptosis via Inhibition of TUBB3 and TOPIIA Gene Expressions in 5637 and K562 Cancer Cell Lines.

OBJECTIVE: Podophyllotoxin (PTOX), a natural compound in numerous plants, contains remarkable biological properties that include anti-tumor, anti-viral such as anti-human im- munodeficiency virus (HIV) activities. In order to avoid its adverse effects, various com- pounds have been derived from PTOX. 6-methoxy PTOX (MPTOX) is one of the natural PTOX derivatives with an extra methoxy group. MPTOX is mostly isolated from the Linum species. This study has sought to determine the biological effects of MPTOX on cancer cell lines, 5637 and K562. MATERIALS AND METHODS: In this experimental study, we treated the 5637 and K562 cancer cell lines with MPTOX in a doseand time-dependent manner. Apoptosis was examined by flow cytometry and viability rate was analyzed by the MTT assay. Expressions of the tubulin (TUBB3) and topoisomerase II (TOPIIA) genes were determined by real-time poly- merase chain reaction (PCR). RESULTS: Treatment with MPTOX led to significant induction of apoptosis in cancer cells compared to control cells. Gene expression analysis showed reduced levels of TUBB3 and TOPIIA mRNA following MPTOX treatment. CONCLUSION: MPTOX inhibited TUBB3 and TOPIIA gene expression and subsequently induced cell death through apoptosis. These results suggested that MPTOX could be considered a potential anti-tumor agent.