Phytohemagglutinin from Phaseolus vulgaris enhances the lung cancer cell chemotherapy sensitivity by changing cell membrane permeability.

Chemotherapy is still a prevalent strategy for clinical lung cancer treatment. However, the inevitable emerged drug resistance has become a great hurdle to therapeutic effect. Studies have demonstrated that the primary cause of drug resistance is a decrease in the chemotherapeutic medicine concentration. Several lectins have been confirmed to be effective as chemotherapy adjuvants, enhancing the anti-tumor effects of chemotherapy drugs. Here, we combined phytohemagglutinin (PHA), which has been reported possess anti-tumor effects, with chemotherapy drugs Cisplatin (DDP) and Adriamycin (ADM) on lung cancer cells to detect the sensitivities of PHA as a chemotherapy adjuvant. Our results demonstrated that the PHA significantly enhanced the sensitivity of lung cancer cells to DDP and ADM, and Western blot showed that PHA combined with DDP or ADM enhance cytotoxic effects by inhibiting autophagy and promoting apoptosis. More importantly, we found PHA enhanced the chemotherapeutic drugs cytotoxicity by changing the cell membrane to increase the intracellular chemotherapeutic drugs concentration. Besides, the combination of PHA and ADM increased the ADM concentration in the multidrug-resistant strain A549-R cells and achieved the drug sensitization effect. Our results suggest that PHA combined with chemotherapy can be applied in the treatment of lung cancer cells and lung cancer multidrug-resistant strains, and provide a novel strategy for clinical tumor chemotherapy and a new idea to solve the problem of drug resistance in clinical lung cancer.

Nicotinic acid modulates microglial TREM-2 gene in Phytohaemagglutinin-Induced in vitro model of Alzheimer's disease like pathology.

Alzheimer's disease (AD) is a multifactorial,neurodegenerative disorder linked withextracellular amyloid beta (Aß) plaques deposition and formation of intracellular neurofibrillary tangles (NFTs). Currently, no effective therapies are available to cure AD. Neuroinflammation isa well-known hallmark in the onset and advancement of AD and triggering receptor expressed on myeloid cells-2 (TREM-2), a microglial gene, is responsible for regulating inflammatory responses and clearance of cellular debris. Loss of TREM-2functionincreases neuroinflammation associated expression of pro-inflammatory markersthus resultingin reduced clearance of Aß that further aid in disease progression.Therefore, targeting neuroinflammation is a good therapeutic approach for AD. This study aimed to determine the neuroprotective effect of nicotinic acid (NA) in vitro model of AD-like pathology induced in F-98 cell line using Phytohemagglutinin (PHA). MTT assay was employed for checking the cell viability as well as the proliferation of the cells following treatment with NA. PHA at the concentration of 10 µg/mL produces maximum plaques. The neuroprotective effect of NA was next evaluated against PHA-induced plaques and it was observed that NA reverses the damages induced by PHA i.e., by inhibiting the clustering of the cells and replacing the damaged cells with the new ones. Further, NA also increased the expression of TREM-2/DAP-12 with parallel decreased in the expression of IL-1ß, TNF-α and iNOS. It also successfully altered disease associated ADAM-10 and BACE-1 compared to PHA control. These findings suggest that NA might be considered as a good therapeutic candidate for the treatment of neurodegenerative disorders like AD.

Phytohemagglutinin ameliorates HFD-induced obesity by increasing energy expenditure.

Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds, and to verify whether they can reduce the body weight gain and investigate their molecular mechanisms. By using drug-screening methods, Phytohemagglutinin (PHA) was found to be the most anti-obesity candidate natural compound. Six-week-old C57BL/6J mice were fed with a high-fat diet (HFD) and intraperitoneally injected with 0.25 mg/kg PHA everyday for 8 weeks. The body weight, glucose homeostasis, oxygen consumption and physical activity were assessed. We also measured the heat intensity, body temperature and the gene expression of key regulators of energy expenditure. Prevention study results showed PHA treatment not only reduced the body weight gain but also maintained glucose homeostasis in HFD-fed mice. Further study indicated energy expenditure and uncoupling protein 1 (UCP-1) expression of brown adipose tissue (BAT) and white adipose tissue (WAT) in HFD-fed mice were significantly improved by PHA. In the therapeutic study, a similar effect was observed. PHA inhibited lipid droplet formation and upregulated mitochondrial-related gene expression during adipogenesis in vitro. UCP-1 KO mice displayed no differences in body weight, glucose homeostasis and core body temperature between PHA and control groups. Our results suggest that PHA prevent and treat obesity by increasing energy expenditure through upregulation of BAT thermogenesis.

T-cell response to phytohemagglutinin in the interferon-γ release assay as a potential biomarker for the response to immune checkpoint inhibitors in patients with non-small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitors are a standard treatment for advanced lung cancer, although it remains important to identify biomarkers that can accurately predict treatment response. Immune checkpoint inhibitors enhance the antitumor T-cell response, and interferon-γ plays an important role in this process. Therefore, this study evaluated whether the number of interferon-γ-releasing peripheral T cells after phytohemagglutinin stimulation in the interferon-γ release assay might act as a biomarker for the response of non-small cell lung cancer to immune checkpoint inhibitor treatment. METHODS: Data were retrospectively collected regarding 74 patients with non-small cell lung cancer who had received immune checkpoint inhibitors. Pretreatment screening tests had been performed using the T-SPOT.TB assay, which quantifies the number of interferon-γ-releasing T cells (as immunospots) in response to phytohemagglutinin and tuberculosis-specific antigen stimulation. Clinical factors and the number of spots in the T-SPOT fields were evaluated for associations with patient outcomes. The median number of spots was used to categorize patients as having high or low values, and the two groups were compared. RESULTS: Relative to patients with a low ratio, patients with a high ratio of phytohemagglutinin/tuberculosis-specific antigen spots (i.e. more responsive T cells) had significantly better progression-free survival after immune checkpoint inhibitor treatment. When we only considered patients with negative T-SPOT results, a high number of phytohemagglutinin-stimulated spots corresponded to significantly longer progression-free survival. CONCLUSION: The T-SPOT.TB assay can be used to quantify the number of immunospots in response to antigen stimulation, which may predict the response to immune checkpoint inhibitors in patients with non-small cell lung cancer.

Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms.

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.

Direct anthelmintic and immunostimulatory effects of oral dosing semi-purified phytohaemagglutinin lectin in sheep infected with Teladorsagia circumcincta and Trichostrongylus colubriformis.

Lectins are plant secondary compounds that can have anthelmintic properties in vitro. In particular, the phytohaemagglutinin lectin extracted from Phaseolus vulgaris has been shown to inhibit the feeding of Trichostrongylus colubriformis and Teladorsagia circumcincta L1 larvae. However, little is known about the potential anthelmintic properties of this lectin in vivo and its suitability to control gastrointestinal parasite infections in lambs. In a 2 × 2 study, lambs were either orally dosed, or not, with 2.3mg semi-purified PHA lectin per kg live weight (LW) per day, whilst concurrently infected, or not, with 1000 T. circumcincta and 1000 T. colubriformis L3 infective larvae per day for 42 days. There were no adverse clinical effects observed with this dose of PHA lectin. Although worm burdens were similar, animals dosed with PHA lectin had reduced concentration of nematode eggs in the faeces compared with their non-lectin dosed counterparts (P=0.026), suggesting that there may be a direct effect of PHA lectin on parasite fecundity. Irrespective of infection, PHA lectin had immune-stimulatory properties with increased eosinophillia in both abomasal and small intestine tissue sections taken at slaughter on day 42 (P<0.02 for both) and a tendency for decreased ability of Teladorsagia larvae to penetrate abomasal tissue explants (P=0.06). Compared with infection alone, concurrent PHA lectin dosing and infection further increased the number of eosinophils (P<0.01), PAS-positive (mucin-producing cells) (P=0.03) and tended to increase the number of T helper cells (P=0.06). No interactions were observed for cell populations in small intestine tissue sections. These results suggest PHA lectin could have two possible modes of action against T. circumcincta and T. colubriformis, a direct anthelmintic effect on nematode fecundity and an indirect effect through enhancing local immune responses in the host.

Cross-linking enhances deposition of human endothelial progenitor cells in the rat heart after intracoronary transplantation.

Transplantation of human endothelial progenitor cells (hEPCs) may improve vascularization and left ventricular function after myocardial infarction. The scope of this study was to explore, whether cross-linking of EPCs may enhance the deposition of cells in the rat heart after clinical-like, intracoronary transplantation. To this end, (111)In-oxinate-labeled hEPCs were infused by a minimally invasive technique into the coronary arteries of immunosuppressed Wistar rats under control conditions and after ischemia/reperfusion. In a second set of experiments hEPCs were treated with phytohemagglutinin to create small cell clusters prior to transplantation. Continous three-dimensional HiSPECT images for 1 h and after 48 h revealed that cell deposition was significantly higher when hEPCs were cross-linked. Therefore, cross-linking of hEPCs may provide a promising approach to enhance the number of trapped cells also in a clinical setting.

Treatment of oral mucositis after peripheral blood SCT with ATL-104 mouthwash: results from a randomized, double-blind, placebo-controlled trial.

ATL-104 is a potent mitogen for epithelial cells of the gastrointestinal tract. In animal models, ATL-104 aids regeneration of the gastrointestinal tract after treatment with chemotherapeutic agents. The effect of ATL-104 on mucositis in patients requiring high-dose melphalan or BEAM before peripheral blood SCT (PBSCT) was investigated in a randomized, placebo-controlled, double-blind, two-part study. Patients were randomized to ATL-104 (50, 100 or 150 mg) or placebo once daily for 3 days before chemotherapy and 3 days after PBSCT. Part one of the study was a dose-escalation design; part two was a parallel group design using all three ATL-104 doses. Patients were followed up for 28 days post-treatment. Severity of signs and symptoms were assessed and used to calculate scores for standard toxicity rating scales (WHO, Western Consortium for Cancer Nursing Research (WCCNR)). Sixty-three patients were treated. Treatment with ATL-104 substantially reduced the median duration of severe oral mucositis (WHO grade 3 or 4) compared with placebo (median duration: ATL-104 groups 2 or 3 days, placebo 10.5 days). The effect of ATL-104 on the incidence of severe oral mucositis was inconclusive. Similar results were obtained using the WCCNR Scale. Adverse events (AEs) were predominantly mild or moderate in intensity. Gastrointestinal AE were most common.

Reducing the availability of polyamines for a developing tumour.

To sustain growth and support metabolic requirements, mammals assimilate energy-producing molecules and nutrients from food. These molecules are distributed throughout the body in order to meet the requirements of the internal organs. The various demands of the different organs are to a large extent met by regulatory processes consisting of a complex interaction between hormones, growth factors and cytokines. Normal metabolic activity and partitioning of nutrients between individual organs is affected by a number of events such as stress, a limited supply of nutrients, infection or tumour growth. Since the intestine has the highest metabolic activity of all the internal organs, a tumour will initially compete with the gut for nutrients and energy-providing molecules. The polyamines represent a class of molecules where the demand in the body increases during tumour growth. A tumour can partly obtain the polyamines required to support its growth by up-regulating its own biosynthetic capacity and partly by increasing uptake from the body pool. Rather than limiting the exogenous supply of dietary polyamines we have used another approach to manipulate polyamine pools in mice. When the lectin phytohaemagglutinin is included in the diet, a fully reversible dose-dependent growth of the small intestine occurs leading to an extensive accumulation of polyamines in the intestinal epithelia. This approach of reducing the availability of exogenous polyamines to a growing tumour will be discussed.

Phytohemagglutinin inhibits lymphoid tumor growth in vitro and in vivo.

Cure rates for a variety of leukemias and lymphomas have improved dramatically over the past several decades, but relapsed disease continues to account for thousands of deaths per year. Viable treatment options for relapsed disease are few, encouraging the development of novel therapies. In the present paper, we describe phytohemagglutinin (PHA), a standard T cell mitogen, as an inhibitor of both T- and B-cell tumors. In vitro studies show that PHA can inhibit incorporation of 3H-thymidine and mediate apoptosis of B- and T-cell tumor lines. The inhibitory effects are enhanced when PHA is used in conjunction with the cell cycle directed drug 5-fluorouracil (5-FU). Phytohemagglutinin treatments can also impede tumor growth in mice while showing no toxic side effects in this animal model.

The effects of lectins on indomethacin-induced small intestinal ulceration.

Growth factors, such as epidermal growth factor and keratinocyte growth factor, have considerable therapeutic potential for repairing mucosal injury in the intestine when given systemically. Recently, several lectins have been shown to have trophic effects on the intestine when given orally. We examined the effects of phytohaemagglutinin (PHA) and concanavalin A (Con-A) on indomethacin-induced intestinal injury in rat. Five-week-old rats were randomized to four groups (n=5), and intestinal injury was induced by indomethacin injection in three of these groups. Elemental diet (ED) feeding was then commenced. The groups were thus ED feeding/indomethacin untreated (control group), ED feeding/indomethacin treated (ED group), 0.1% PHA-supplemented ED feeding/indomethacin treated (PHA group) and 0.1% Con-A-supplemented ED feeding/indomethacin treated (Con-A group). After 7 days of feeding, macroscopic inflammatory scores, mucosal permeability, myeloperoxidase (MPO) activities and cell proliferation were determined. Macroscopic inflammatory scores, mucosal permeability and MPO activities were significantly lower in both lectin groups than that in control group. Twenty-four hour excretion rate of phenolsulphonphthalein was significantly lower in both lectin groups than that in ED group. Cell proliferation of the small intestine was significantly increased by both lectins. Lectin supplementation can induce ulcer healing following indomethacin-induced damage.

A comparison of the lymphoproliferative capacity of pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) in CCR-5 wild-type and heterozygous HIV-infected and uninfected subjects.

Effect of CCR-5 delta 32 heterozygosity in immunological protection was studied by a lymphocyte proliferation assay. Twenty of 86 HIV+ and eight of 32 healthy subjects showed heterozygous mutation (wt/mut) of the CCR-5 gene. Lymphocyte proliferation to pokeweed mitogen was found significantly higher (P < 0.005) in wt/mut versus wild type homozygous (wt/wt) HIV+ subjects in groups with CD4 > 500 and CD4 < 200 cell/ micro L. Phytohaemagglutinin induced stronger proliferation of cells from wt/mut HIV+ subjects with CD4 < 200 cell/ micro L (P = 0.03). Decline of lymphocyte response was more significant among wt/wt groups with different CD4+ cell counts than that between wt/mut groups to both mitogens. Reduced number of CCR-5 receptors on CD4+ cells may decrease the ability of HIV-1 envelope glycoproteins to transduce intracellular signals through CCR-5. Mutation in CCR-5 gene seems to have a benefit in preventing T-cells from HIV envelope-mediated immunopathogenic effects and maintain a relatively normal response to lectins.

Cyclins D1, E, A expression and synergistic cytotoxicity to a cholangiocarcinoma cell line from recombinant TNF-alpha and PHA supernate.

We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.

Mitogens for curative therapy of HIV-1 infections; an update.

The curative objectives of administering mitogens in conjunction with HAART have been to initiate killer reactions against HIV-1, replenish CD4 cells, rejuvenate CD8 cells, stimulate effective immune reactions against malignancies, generate tolerated immune responses against conventional and opportunistic infections, resist treatment-evading tactics of HIV mutations, and reconstitute both immune and hematopoietic competences. With recent observations that these aims might be thwarted by sequestration of HIV-1 infection in memory cells residing in the circulation, lymph nodes, and spleen as well as in various other sites not readily accessible to eradication, stronger emphasis should be focused primarily on mitogen-induced blockage of HIV-1 entry into CD4 cells. Equally important would be the mitogen panactivation of any cells sequestering virions and proviruses, thereby forcing them out of the latent G0 phase and exposing them to destruction by HAART and the immune responses. While the currently best established mitogens for HIV-1 therapy, PHA-L4 and PWM, might be successfully applied independently or in tandem, other mitogens may be found suitable for evaluation.

Mitogen therapy for biological warfare/terrorist attacks and viral hemorrhagic fever control.

Ken Alibek was for 17 years a leader in Biopreparat, the Soviet Union's top secret agency involved in developing and stockpiling the most lethal bacteria, viruses, and toxins in the history of mankind before he defected with his family to the United States in 1992. Very contrite when he discovered he had been misled to believe that his efforts had been essential to the survival of his homeland, Alibek has become active sounding an alarm about, among other things, thousands of unemployed Russian scientists who have been seeking survival by selling their destructive expertise to rouge states and bioterrorists. Working full time in devising protective measures that might help control the damaging effects of terrorist attacks, Alibek has placed strong emphasis on stimulating nonspecific immunities of victims mainly with interleukins and other cytokines. A more productive alternative would be giving mitogens such as PHA and PWM to reinforce vaccine and antibiotic actions, at the same time stimulating protective immune, myelopoietic, and lymphopoietic responses. A key objective would be to find an effective management for the dreaded viral hemorrhagic fevers. Using Ebola infection as an experimental model, Yang et al. have shown that PHA can block both the viral secretions that inhibit neutrophil immune responses and the viral transmembrane glycoprotein that facilitates damage of the human endothelial cells responsible for the lethal hemorrhagic manifestations. Normal serum glycoproteins have in the past been clearly shown to inhibit the functions of PHA, thereby increasing dosage requirements. Extrapolation of this interaction with serum glycoproteins suggests that PHA given intravenously in adequate dosage should readily be able to block the deleterious Ebola virus glycoprotein effects. Data in an extensive classification of the hemorrhagic fever viruses recently presented by Barry make it possible to predict that mitogen therapy should be effective for virtually all of the disorders included. Therapeutic trials should best start with intravenous administration of PHA since this is the mitogen about which most is known and the only one given to humans, although the nonagglutinating advantages of fraction i.v. of PHA should be evaluated as a replacement. Functioning in a different mode, PWM has the advantage of much greater potency, and can be given either intravenously or orally, since these appear to be equally effective routes of administration. The best means of properly integrating the use of these mitogens needs to be determined.

Attempts of modification of apoptosis in the course of experimental trichinellosis in mice.

The influence of the some immunomodulators (PHA-P, TFX and dexamethasone) on the process of apoptosis, occurring in the course of trichinellosis in mice, has been studied. It has been found that PHA-P activates this process in the jejunum mucosa and prolongs it in the muscular inflammatory infiltration, whereas TFX has no influence and dexamethasone distinctly decreases the level of the apoptotic cells. The number of the intestinal trichinae on the successive days of infection was similar in all groups of animals, however, the number of the muscular larvae in the groups receiving immunostimulators was much lower and in the group treated with dexamethasone--a little higher than that in control. As in mice receiving PHA-P and TFX, the cellular inflammatory infiltration in the muscles was larger than that in control, and in the group to which dexamethasone was administrated--smaller, the authors think that it was extensiveness of the infiltration and not the level of the apoptotic cells that influenced the number of the outliving larvae.