Semisynthesis and biological activity of aminoacyl triesters of squamocin, an annonaceous acetogenin

A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata, have been synthesized in two to three steps from protected (l)-aminoacids and squamocin 1 using standard coupling/deprotection procedures. These semisynthetic analogs were tested on submitochondrial particles (SMP) for their complex I inhibitory activities, and against KB 3-1 cells in vitro. All triesters derivatives exhibited a complete extinction of activity at the enzymatic level, correlated to a reduced though modulated cytotoxicity in comparison with squamocin 1. This activity can apparently be considered as a function of the amphipathy of the analogs, the more amphiphilic ones being the more cytotoxic.     

Annonacin,a mono-tetrahydrofuran acetogenin,arrests cancer cells at the G1 phase and causes cytotoxicity in a Bax- and caspase-3-related pathway

Annonaceous acetogenins are a group of potential anti-neoplastic agents isolated from Annonaceae plants. In this study, we purified annonacin, a cytotoxic mono-tetrahydrofuran acetogenin, from the seeds of Annona reticulata and analyzed its biological effects. Herein, we have shown that annonacin caused significant cell death in various cancer cell lines. T24 bladder cancer cells at the S phase were more vulnerable to the cytotoxicity of annonacin. Furthermore, annonacin activated p21 in a p53-independent manner and arrested T24 cells at the G1 phase. It also induced Bax expression, enhanced caspase-3 activity, and caused apoptotic cell death in T24 cells. In summary, these results suggest that annonacin is potentially a promising anti-cancer compound.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Do cancer cells in human and meristematic cells in plant exhibit similar responses toward plant extracts with cytotoxic activities?

We examined the effect of water extracts of Persea americana fruit, and of the leaves of Tabernamontana divericata, Nerium oleander and Annona cherimolia (positive control) on Vicia faba root cells. We had confirmed in our previously published data the cytotoxicity of these plant extracts on four human cancer cell lines: liver (HepG-2), lung (A549), colon (HT-29) and breast (MCF-7). Vicia faba roots were soaked in plant extracts at dilutions of 100, 1,250, 2,500, 5,000, 10,000, 20,000 ppm for 4 and 24 h. All treatments resulted in a significant reduction in the mitotic index in a dose dependant manner. Root cells treated with T. divericata, N. oleander and A. cherimolia exhibited a decrease in prophase cell percentage, increase in micronuclei and chromosomal abnormalities as concentration increased. The P. americana treatment showed the highest cytotoxic effect on cancer cells, prophase cell percentage increased linearly with the applied concentration and no micronuclei were detected. This study shows that root tip assay of beans can be used in initial screening for new plant extracts to validate their use as candidates for containing active cytotoxic agents against malignant cells. This will greatly help in exploring new plant extracts as drugs for cancer treatment.     

Induction of G2/M phase arrest by squamocin in chronic myeloid leukemia (K562) cells

Squamocin is one of the annonaceous acetogenins and has been reported to have anticancer activity. Squamocin was found to inhibit the growth of K562 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in K562 cells following 24 h exposure to squamocin. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a dose-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that squamocin did not change the steady-state levels of Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D3 and cyclin E, but decreased the protein levels of Cdk1 and Cdc25C. These results suggest that squamocin inhibits the proliferation of K562 cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.     

Selective action of acetogenin mitochondrial complex I inhibitors

Five annonaceous acetogenins, rolliniastatin-1 [structure: see text], rolliniastatin-2 [structure: see text], laherradurin [structure: see text], squamocin [structure: see text], annonacin [structure: see text], and rotenone as a reference, differing in their NADH oxidase inhibition activity, have been evaluated for antifeedant, insecticidal, trypanocidal and cytotoxic effects on insect, mammalian and tumor cells. All the test compounds were toxic to Leptinotarsa decemlineata, demonstrated selective cytotoxicity to insect Sf9 cells and a panel of tumor cell lines with the multidrug-resistant SW480 (P-glycoprotein+, Pgp+) being the most sensitive one. Compounds [structure: see text] and rotenone had post-ingestive effects against Spodoptera littoralis larvae while [structure: see text] and rotenone were active against Trypanosoma cruzi. Based on their biochemical properties (inhibition of the mitochondrial NADH oxidase activity), the in vivo effects of these compounds on S. littoralis and their cytotoxic effects on Sf9 and tumor cells were more predictable than their effect on T. cruzi and mammalian cells.     

Bystander elimination of antigen loss variants in established tumors

Cancers express antigens that are targets for specific cytotoxic T lymphocytes (CTLs). However, cancer cells are genetically unstable. Consequently, sub-populations of cancer cells that no longer express the target antigen may escape destruction by CTLs and grow progressively. We show that cytotoxic T cells indirectly eliminate these antigen loss variants (ALVs) in a model system when the parental cancer cells express sufficient antigen to be effectively cross-presented by the tumor stroma. When the parental tumor expressed lower levels of antigen, cytotoxic T cells eradicated the antigen-positive parental cancer cells, but the ALVs escaped, grew and killed the host. By contrast, when the parental tumor expressed higher levels of antigen, cytotoxic T cells eradicated not only the parental cancer cells but also the ALVs. This 'bystander' elimination of ALVs required stromal cells expressing major histocompatibility complex (MHC) molecules capable of presenting the antigen, and occurred in tumors showing evidence of stromal destruction. ALVs were apparently eliminated indirectly when tumor-specific CTLs killed stromal cells that were cross-presenting antigen produced by and released from antigen-positive cancer cells. These results highlight the general importance of targeting the tumor stroma to prevent the escape of variant cancer cells.     

Frequent detection of anti-recoverin cytotoxic T-lymphocyte precursors in peripheral blood of cancer patients by using an HLA-A24-recoverin tetramer

Recoverin is a photoreceptor-specific calcium binding protein that is only expressed in the retina in normal tissues. Aberrant expression of recoverin, however, has been observed in several cancer tissues and may cause a very rare autoimmune disease, cancer-associated retinopathy (CAR). It has been suggested that CAR-positive cancer patients have a more favorable prognosis than CAR-negative cancer patients. To estimate the status of recoverin-specific T cells in such cancer patients, we generated an HLA-A24-recoverin peptide tetramer. By use of the tetramer, we could directly assess the frequency of CTL precursors (CTLp) of 20 HLA-A24(+) cancer patients with ten colon, six stomach and four breast cancers, and seven healthy individuals. Four cancer patients showed a CTLp frequency higher than 0.9%. Seven cancer patients including the former four patients and two healthy individuals showed specific anti-recoverin cytotoxic responses in an HLA-A24-restricted manner after in vitro stimulation with the recoverin peptide. Moreover, five cancer patients analyzed in an independent experiment using different peripheral blood mononuclear cells (PBMC) samples showed similar CTLp and cytotoxic T lymphocyte (CTL) frequencies and cytotoxic responses, suggesting that the CTLp frequency analyzed by the tetramer and the cytotoxic response may have a good correlation. Thus, we hypothesize that anti-recoverin CTLp may exist in some cancer patients, and that anti-recoverin CTL may be readily induced.     

Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow

In an analysis of 84 primary-operated breast cancer patients and 11 healthy donors, we found that the bone marrow of most patients contained memory T cells with specificity for tumor-associated antigens. Patients' bone marrow and peripheral blood contained CD8+ T cells that specifically bound HLA/peptide tetramers. In short-term culture with autologous dendritic cells pre-pulsed with tumor lysates, patients' memory T cells from bone marrow (but not peripheral blood) could be specifically reactivated to interferon-gamma-producing and cytotoxic effector cells. A single transfer of restimulated bone-marrow T cells into NOD/SCID mice caused regression of autologous tumor xenotransplants associated with infiltration by human T cells and tumor-cell apoptosis and necrosis. T cells from peripheral blood showed much lower anti-tumor reactivity. Our findings reveal an innate, specific recognition of breast cancer antigens and point to a possible novel cancer therapy using patients' bone-marrow-derived memory T cells.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

Cardiobutanolide,a styryllactone from Goniothalamus cardiopetalus

A styryllactone namely cardiobutanolide was isolated from the stem bark of Goniothalamus cardiopetalus together with four known styryllactones goniothalamin, goniodiol, goniofufurone, goniofupyrone and known acetogenins squamocin and an epimeric mixture of goniodonin and 34-epi-goniodonin. The structure of the new compound was elucidated on the basis of 1D and 2D NMR experiments and mass spectroscopic techniques.     

Methyl jasmonate induces cell cycle block and cell death in the amitochondriate parasite Trichomonas vaginalis

Jasmonates are a group of small lipids produced in plants and function as stress hormones. They are selectively cytotoxic against cancer cells. Methyl jasmonate (MJ), one of the naturally occurring jasmonates, has direct mitochondriotoxic effects, strongly suggesting that mitochondria are target organelles of jasmonates. We have previously shown that jasmonates are cytotoxic to two human parasites -Schistosoma mansoni and Plasmodium falciparum. Both the cancer cells and the parasites mentioned above possess mitochondria. The present work aimed to examine whether jasmonates are able to damage cells lacking mitochondria, e.g., some unicellular human parasitic flagellates. We found that MJ induced death of the amitochondriate Trichomonas vaginalis parasites. MJ caused fragmentation and condensation of the DNA of T. vaginalis, resembling phenomena associated with apoptotic death. However, DNA laddering, a sub-G(1) cell cycle stage peak and caspase-3 activation were not observed. Thus, MJ-induced T. vaginalis cell death appears to be non-apoptotic. We found that MJ induced cell cycle block at the G(2)/M phase in T. vaginalis, similar to the effect of metronidazole. We examined the influence of MJ on the bioenergetic pathways of T. vaginalis, and found that depletion of ATP did not precede death of the parasites, but rather reflected it. Nevertheless, 2-deoxy-d-glucose, a glycolysis blocker, was synergistic with MJ in causing death of T. vaginalis cells, suggesting that MJ does perturb the bioenergetic homeostasis of the parasites. Finally, MJ was found to be cytotoxic towards a metronidazole-resistant strain of T. vaginalis (ATCC 50143), suggesting that it may be effective for the treatment of nitroimidazole-refractory trichomoniasis. In conclusion, MJ was found to exhibit mitochondria-independent cytotoxicity and presents a potentially novel agent against T. vaginalis.     

Bufalin induces G(0)/G(1) phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells

Most of the chemotherapy treatments for bladder cancer aim to kill the cancer cells, but a high recurrence rate after medical treatments is still occurred. Bufalin from the skin and parotid venom glands of toad has been shown to induce apoptotic cell death in many types of cancer cell lines. However, there is no report addressing that bufalin induced cell death in human bladder cancer cells. The purpose of this study was investigated the mechanisms of bufalin-induced apoptosis in a human bladder cancer cell line (T24). We demonstrated the effects of bufalin on the cell growth and apoptosis in T24 cells by using DAPI/TUNEL double staining, a PI exclusion and flow cytometric analysis. The effects of bufalin on the production of reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔΨ(m)), and DNA content including sub-G1 (apoptosis) in T24 cells were also determined by flow cytometry. Western blot analysis was used to examine the expression of G(0)/G(1) phase-regulated and apoptosis-associated protein levels in bufalin-treated T24 cells. The results indicated that bufalin significantly decreased the percentage of viability, induced the G(0)/G(1) phase arrest and triggered apoptosis in T24 cells. The down-regulation of the protein levels for cyclin D, CDK4, cyclin E, CDK2, phospho-Rb, phospho-AKT and Bcl-2 with the simultaneous up-regulation of the cytochrome c, Apaf-1, AIF, caspase-3, -7 and -9 and Bax protein expressions and caspase activities were observed in T24 cells after bufalin treatment. Based on our results, bufalin induces apoptotic cell death in T24 cells through suppressing AKT activity and anti-apoptotic Bcl-2 protein as well as inducing pro-apoptotic Bax protein. The levels of caspase-3, -7 and -9 are also mediated apoptosis in bufalin-treated T24 cells. Therefore, bufalin might be used as a therapeutic agent for the treatment of human bladder cancer in the future.     

Qualitative HPLC‐DAD/ESI‐TOF‐MS Analysis,Cytotoxic, and Apoptotic Effects of Croatian Endemic Centaurea ragusina L. Aqueous Extracts

Centaurea ragusina L., an endemic Croatian plant species, revealed a good cytotoxic activity of aqueous extracts (AE) on human bladder (T24) and human glioblastoma (A1235) cancer cell lines. The chemical constituents were tentatively identified using high performance liquid chromatography HPLC‐DAD/ESI‐TOF‐MS in negative ionization mode. The main compounds of herba extract were sesquiterpene lactones: solstitialin A 3,13‐diacetate and epoxyrepdiolide; organic acid: quinic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, and malic acid; sesquiterpene lactone: cynaropicrin; phenolic compounds: chlorogenic acid and phenylpropanoid: syringin. The AE of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastoma (A1235) cancer cell lines using the MTT assay. Although both extracts showed significant dose‐ and time‐dependent cytotoxic activity against both cancer cell lines, the flower extract exhibited slightly higher activity. In order to determine type of cell death induced by treatment, cell lines were exposed subsequently to a treatment with both flower and herba AE. The majority of the cells died by induced apoptosis treatment. Flower AE (26.25%), compared to a leaf AE (22.15%) showed slightly higher percentage of an apoptosis in T24 cells, when compared to a non‐treated cells (0.04%).     

Aromin-A, an Annonaceous acetogenin from Annona cherimola.

Chromatographic fractionation of a MeOH extract from the stems of Annona cherimola led to the isolation of a new non-adjacent, bis-THF ring acetogenin (one THF ring being at C-4 and the other at C-16), aromin-A (1) along with the known cytotoxic acetogenin squamocin (2). The structures of these isolates were established by means of mass spectrometry and spectroscopic techniques.     

The effects of neuraminidase and galactose oxidase on murine lymphocytes. I. Evidence for the differential delivery of signal(s) leading to cell proliferation and the differentiation of cytotoxic T cells.

The sequential treatment of normal C57BL/6 mouse spleen cell populations with neuraminidase (NA) and galactose oxidase (GO) resulted in cell proliferation, but not in the differentiation of cytotoxic T cells. In contrast, C57BL/6 spleen cells derived from animals primed 5 to 8 months earlier with alloantigen (P815 mastocytoma cells of the DBA/2 strain) both proliferated and demonstrated T cell-mediated cytotoxicity after NAGO stimulation. T cells differentiating into cytotoxic cells after NAGO treatment demonstrated properties similar to alloantigen-specific 'memory' T cells. These were: 1) cytotoxicity developed only from 'primed' cell populations, 2) cytotoxicity developed within 24 hr after NAGO treatment, 3) DNA synthesis was not required for the differentiation of cytotoxic cells during the first 24 hr of culture but both DNA synthesis and cell proliferation were required for the cytotoxicity developing after 24 hr, and 4) all cytotoxicity induced by NAGO showed specificity for the priming alloantigen. It was found, furthermore, that cytotoxicity could be induced at much lower GO concentrations than needed for increased DNA synthesis. We interpret this finding as an indication that NAGO can differentially deliver two 'signals' to T lymphocytes: one leading to cell proliferation, the other causing the differentiation of memory T cells into cytotoxic effectors.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

Efficient tumor cell lysis mediated by a Bcl-X(L) specific T cell clone isolated from a breast cancer patient

Based on the detection of spontaneous immune responses in cancer patients with cancer of different origin, Bcl-X(L) was recently described as a highly interesting tumor antigen recognized by CD8 positive cytotoxic T lymphocytes. To further characterize Bcl-X(L) as a tumor antigen we isolated and expanded Bcl-X(L) specific T cells from the peripheral blood of a breast cancer patient hosting a strong Bcl-X(L) specific T cell response. We describe that HLA-A2 restricted Bcl-X(L) specific T cell clones very efficiently lyse peptide pulsed T2 cells. Furthermore, tumor cell lines of different origin, i.e., breast cancer, colon cancer, and melanoma, are efficiently lysed in an HLA-dependent manner. Finally, ex vivo-isolated leukemia cells, but not non-malignant B and T cells are killed by Bcl-X(L) specific T cells. Our data underline Bcl-X(L) as an universal tumor antigen widely applicable in specific anticancer immunotherapy.     

Recent developments in cancer vaccines

The adoptive transfer of cancer Ag-specific effector T cells in patients can result in tumor rejection, thereby illustrating the immune system potential for cancer therapy. Ideally, one would like to directly induce efficient tumor-specific effector and memory T cells through vaccination. Therapeutic vaccines have two objectives: priming Ag-specific T cells and reprogramming memory T cells (i.e., a transformation from one type of immunity to another, for example, regulatory to cytotoxic). Recent successful phase III clinical trials showing benefit to the patients revived cancer vaccines. Dendritic cells (DCs) are essential in generation of immune responses, and as such represent targets and vectors for vaccination. We have learned that different DC subsets elicit different T cells. Similarly, different activation methods result in DCs able to elicit distinct T cells. We contend that a careful manipulation of activated DCs will allow cancer immunotherapists to produce the next generation of highly efficient cancer vaccines.