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Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein-Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

Reactivation of Epstein-Barr virus (EBV) is associated with EBV-associated malignancies and is considered to be a benefit target for treatment. Andrographolide is claimed to have antiviral and anti-tumor activities. Therefore, this study aimed to investigate the effect of andrographolide on the inhibition of EBV lytic reactivation in EBV-positive cancer cells. The cytotoxicity of andrographolide was firstly evaluated in EBV-positive cancer cells; P3HR1, AGS-EBV and HONE1-EBV cells, using an MTT assay. Herein, the spontaneous expression of EBV lytic genes; BALF5, BRLF1 and BZLF1, was significantly inhibited in andrographolide-treated cells. Accordingly, andrographolide inhibited the expression of Zta and viral production in sodium butyrate (NaB)-induced EBV lytic reactivation. Additionally, proteomics and bioinformatics analysis revealed the differentially expressed proteins that inhibit EBV lytic reactivation in all treated cell lines were functionally related with the histone modifications and chromatin organization, such as histone H3-K9 modification and histone H3-K27 methylation. Taken together, andrographolide inhibits EBV reactivation in EBV-positive cancer cells by inhibiting EBV lytic genes, probably, through the histone modifications.

Background: In our previous study, the semi-synthetic analog of andrographolide, 3,19-isopropylideneandrographolide (IPAD), acts more effectively against herpes simplex virus (HSV) infection in cell culture than does acyclovir. IPAD inhibits cytopathic effect and production of HSV wild types and drug-resistant strains. Its effect is associated with the reduction of immediate-early regulatory protein (ICP27) and early proteins (ICP8 and UL42), indicating a mode of action different from that of acyclovir. Therefore, studies of the anti-HSV activity of IPAD in animal models are required before further application., Material & Method: Prednisolone-treated BALB/c mice were cutaneously infected with HSV-1 wild-type KOS strain. Experimental groups included control groups (untreated or treated only with the cream base) and treatment groups (with acyclovir or IPAD creams). Creams were applied four times daily for 10 days after infection to the relevant groups. The skin lesion score was assessed twice a day for 10 days. In addition, the effect of IPAD on HSV copy number and HSV late gene (gD) expression was investigated in skin lesion cells by quantitative real-time polymerase chain reaction., Result: IPAD cream was significantly effective in delaying the development of skin lesions and regression of the skin lesion score by day 5 (P < 0.01) compared with untreated controls. In addition, this IPAD cream significantly reduced HSV DNA copy number and gD gene expression (P < 0.01). No signs of irritation were observed at the application site., Conclusion: Topical administration of IPAD cream reduced skin lesions in mice cutaneously infected with HSV-1 KOS.

Andrographolide is the principal bioactive chemical constituent of Andrographis paniculata and exhibits activity against several viruses, including Epstein-Barr virus (EBV). However, the particular mechanism by which andrographolide exerts an anti-EBV effect in EBV-associated gastric cancer (EBVaGC) cells remains unclear. We investigated the molecular mechanism by which andrographolide inhibits lytic reactivation of EBV in EBVaGC cells (AGS-EBV cell line) using proteomics and bioinformatics approaches. An andrographolide treatment altered EBV protein-expression patterns in AGS-EBV cells by suppressing the expression of EBV lytic protein. Interestingly cellular transcription factors (TFs), activators for EBV lytic reactivation, such as MEF2D and SP1, were significantly abolished in AGS-EBV cells treated with andrographolide and sodium butyrate (NaB) compared with NaB-treated cells. In contrast, the suppressors of EBV lytic reactivation, such as EZH2 and HDAC6, were significantly up-regulated in cells treated with both andrographolide and NaB compared with NaB treatment alone. In addition, bioinformatics predicted that HDAC6 could interact directly with MEF2D and SP1. Furthermore, andrographolide significantly induced cell cytotoxicity and apoptosis of AGS-EBV cells by induction of apoptosis-related protein expression. Our results suggest that andrographolide inhibits EBV lytic reactivation by inhibition of host TFs, partially through the interaction of HDAC6 with TFs, and induces apoptosis of EBVaGC cells.

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.

A diterpenoid lactone, 3,19-isopropylideneandrographolide (IPAD) compound isolated from Andrographis paniculata (Burm. f.) Nees, has been reported to inhibit herpes simplex virus type 1 (HSV-1) infection at the post-entry step. To identify the molecular target of IPAD, this study characterized the inhibitory effect of IPAD on infection of Vero cells by HSV-1, HSV-2 and a drug-resistant (DR) HSV-1 strain ACGr4 (acyclovir-resistant and thymidine kinase (TK)-deficient). Viral production, gene and protein expression were determined using plaque assays, quantitative RT-PCR and western blotting, respectively. The results showed that IPAD inhibited HSV-1, HSV-2 and DR-HSV-1 infections at 6-12 h post-infection, a time that corresponded with E gene expression. IPAD completely suppressed ICP8 transcription and translation as well as DNA replication and gD expression in the three strains tested, while acyclovir suppressed transcription and translation of UL30 and gD of HSV-2, HSV-1, but had no effect on DR-HSV-1. These results showed that IPAD has a different molecular target from acyclovir and might therefore be an alternative drug for HSV-1 and HSV-2 wild types and DR-HSV-1 strains., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: An andrographolide analogue, 3, 19-isopropylideneandrographolide (IPAD), exerts an inhibitory effect on replication of wild-type herpes simplex virus serotype 1 (HSV-1). In this study, we examined the anti-viral activity of IPAD on HSV wild types (HSV-1 strain KOS and HSV-2 clinical isolate) and HSV-1 drug-resistant strains (DRs). Synergistic effects of IPAD with acyclovir (ACV) were also evaluated., Methods: MTT and cytopathic effect (CPE) reduction assays were performed to determine cytotoxicity and anti-viral activities, respectively. A combination assay was used to determine synergistic effects of IPAD and ACV. Presence of viral DNA and protein in experimental cells was investigated using the polymerase chain reaction and western blotting, respectively., Results: A non-cytotoxic concentration of IPAD (20.50 μM) completely inhibited CPE formation induced by HSV wild types and HSV-1 DRs after viral entry into the cells. The anti-HSV activities included inhibition of viral DNA and protein synthesis. The minimum inhibitory concentrations of ACV for HSV wild types and HSV-1 DRs were 20.20 and 2,220.00 μM, respectively. Combination of ACV with IPAD showed synergistic effects in inhibition of CPE formation, viral DNA and protein synthesis by HSV wild types as well as HSV-1 DRs. For the synergistic effects on HSV wild types and HSV-1 DRs, the effective concentrations of ACV were reduced., Conclusions: These results showed the inhibitory potential of IPAD on HSV wild types and HSV-1 DRs and suggested that IPAD could be used in combination with ACV for treatment of HSV-1 DRs infections.

Andrographolide, an ENT-labdane diterpene, has been found to have activities against many viruses. Three free hydroxyls at C-3, C-14, and C-19 are involved in the activities. No stage of action has ever been explored. In this study, the naturally occurring compounds of andrographolide, 14-deoxy-11,12-didehydroandrographolide and 14-deoxyandrographolide, and eight semisynthetic analogues, modified at the three free OHs of andrographolide, were explored for their anti-HSV-1 activities. The concentrations that produced 80 % viable cells were used to test for both pre- and postinfections by using cytopathic effect reduction assays on Vero cell cultures. Three analogues, 14-acetyl-3,19-isopropylideneandrographolide, 14-acetylandrographolide, and 3,14,19-triacetylandrographolide, significantly exhibited preinfection step activity against the virus. For postinfection activity, only 3,19-isopropylideneandrographolide showed absolute inhibition of HSV-1 replication. Meanwhile, andrographolide exhibited slight inhibitory activities of 34.48 ± 6.93 % and 56.90 ± 2.65 % against HSV-1 for pre- and postinfection, respectively. The results confirm that the three hydroxyl moieties play a role in the anti-HSV-1 activity of andrographolide. From the study, it can be concluded that 14-acetyl analogues are good for blocking the viral entry, and 3,19-isopropylideneandrographolide, a cyclic dioxane analogue, is good for exerting postinfection anti-HSV-1 activity., (© Georg Thieme Verlag KG Stuttgart · New York.)

Andrographolide (1) and 14-deoxy-11,12-didehydroandrographolide (2) are active constituents of Andrographis paniculata (Burm. f.), family Acanthaceae. A. paniculata extracts are reported to have antiviral, antipyretic, immunostimulant and anticancer activities. In this study, 1 and its 14-acetyl- (4) and 3,19-isopropylidenyl- (3) derivatives, as well as 2 and its 3,19-dipalmitoyl-derivative (5), were intraperitoneally tested for their analgesic, antipyretic, anti-inflammatory and acute toxicity effects in animal models. Analgesic effects were tested in mice using hot plate and writhing tests to distinguish the central and peripheral effects, respectively. The results showed that, at 4 mg/kg, all tested substances have significant analgesic effects, and the highest potency was seen with 3, 4 and 5. Increasing the dose of 3 and 5 to 8 mg/kg did not increase the analgesic effect. In the writhing test, 3 and 5, but not 1, showed significant results. In a baker's yeast-induced fever model, 3 and 5 significantly reduced rats' rectal temperature (p < 0.05). In a carrageenan-induced inflammation model, 1, 3 and 5 significantly reduced rats' paw volume. Doses of 3 and 5 up to 100 mg/kg did not show any serious toxic effects. From this study, 3 and 5 are the most interesting derivatives, showing much greater potency than their parent compounds. These could be further developed as analgesic, antipyretic and anti-inflammatory agents, without any serious toxicity.