Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes.

The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

An improved preparation of phorbol from croton oil.

Background: Croton oil is the only commercial source of the diterpenoid phorbol (1a), the starting material for the semi-synthesis of various diesters extensively used in biomedical research to investigate cell function and to evaluate in vivo anti-inflammatory activity. While efficient chemoselective esterification protocols have been developed for phorbol, its isolation from croton oil is technically complicated, and involves extensive manipulation of very toxic materials like the oil or its native diterpenoid fraction. Results: The preparation of a crude non-irritant phorboid mixture from croton oil was telescoped to only five operational steps, and phorbol could then be purified by gravity column chromatography and crystallization. Evidence is provided that two distinct phorboid chemotypes of croton oil exist, differing in the relative proportion of type-A and type-B esters and showing different stability to deacylation. Conclusion: The isolation of phorbol from croton oil is dangerous because of the toxic properties of the oil, poorly reproducible because of differences in its phorboid profile, and time-consuming because of the capricious final crystallization step. A solution for these issues is provided, suggesting that the poor-reproducibility of croton oil-based anti-inflammatory assays are the result of poor quality and/or inconsistent composition of croton oil.

The reaction of cinnamaldehyde and cinnam(o)yl derivatives with thiols.

Spurred by the alleged relevance of the thia-Michael reaction in the bioactivity of various classes of cinnam(o)yl natural products and by the development of a quick NMR assay to study this reaction, we have carried out a systematic study of the "native" reactivity of these compounds with dodecanethiol and cysteamine as models, respectively, of simple thiols and reactive protein thiols that can benefit from iminium ion catalysis in Michael reactions. Cinnamoyl esters and amides, as well as cinnamyl ketones and oximes, did not show any reactivity with the two probe thiols, while cinnamaldehyde (1a) reacted with cysteamine to afford a mixture of a thiazoline derivative and compounds of multiple addition, and with aliphatic thiols to give a single bis-dithioacetal (6). Chalchones and their vinylogous C5-curcuminoid derivatives were the only cinnamoyl derivatives that gave a thia-Michael reaction. From a mechanistic standpoint, loss of conjugation in the adduct might underlie the lack of a native Michael reactivity. This property is restored by the presence of another conjugating group on the carbonyl, as in chalcones and C5-curcuminoids. A critical mechanistic revision of the chemical and biomedical literature on cinnamaldehyde and related compounds seems therefore required.

Determination of free and glucosidically-bound volatiles in plants. Two case studies: L-menthol in peppermint (Mentha x piperita L.) and eugenol in clove (Syzygium aromaticum (L.) Merr. & L.M.Perry).

This study arises from both the today's trend towards exploiting plant resources exhaustively, and the wide quantitative discrepancy between the amounts of commercially-valuable markers in aromatic plants and those recovered from the related essential oil. The study addresses the determination of both the qualitative composition and the exhaustive distribution of free and glucosidically-bound L-menthol in peppermint aerial parts (Mentha x piperita L., Lamiaceae) and of eugenol in dried cloves (Syzygium aromaticum (L.) Merr. & L.M.Perry, Myrtaceae), two plants known to provide widely ranging essential oil yields. The two markers were investigated in essential oils and residual hydrodistillation waters, before and after enzymatic hydrolysis. Their amounts were related to those in the headspace taken as reference. The results showed that the difference between marker compound in headspace and in essential oil amounted to 22.8% for L-menthol in peppermint, and 16.5% for eugenol in cloves. The aglycones solubilised in the residual hydrodistillation waters were 7.2% of the headspace reference amount for L-menthol, and 13.3% for eugenol, respectively representing 9.3% and 15.9% of their amounts in the essential oil. The amount of L-menthol from its glucoside in residual hydrodistillation waters was 20.6% of that in the related essential oil, while eugenol from its glucoside accounted for 7.7% of the amount in clove essential oil. The yield of L-menthol, after submitting the plant material to enzymatic hydrolysis before hydrodistillation, increased by 23.1%, and for eugenol the increase was 8.1%, compared to the amount in the respective conventional essential oils. This study also aimed to evaluate the reliability of recently-introduced techniques that are little applied, if at all, in this field. The simultaneous use of high-concentration-capacity sample preparation techniques (SBSE, and HS-SPME and in-solution SPME) to run quali-quantitative analysis without sample manipulation, and direct LC-MS glucoside analysis, provided cross-validation of the results.

A cannabigerol quinone alleviates neuroinflammation in a chronic model of multiple sclerosis.

Phytocannabinoids like ∆(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) show a beneficial effect on neuroinflammatory and neurodegenerative processes through cell membrane cannabinoid receptor (CBr)-dependent and -independent mechanisms. Natural and synthetic cannabinoids also target the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ), an attractive molecular target for the treatment of neuroinflammation. As part of a study on the SAR of phytocannabinoids, we have investigated the effect of the oxidation modification in the resorcinol moiety of cannabigerol (CBG) on CB(1), CB(2) and PPARγ binding affinities, identifying cannabigerol quinone (VCE-003) as a potent anti-inflammatory agent. VCE-003 protected neuronal cells from excitotoxicity, activated PPARγ transcriptional activity and inhibited the release of pro-inflammatory mediators in LPS-stimulated microglial cells. Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS) was used to investigate the anti-inflammatory activity of this compound in vivo. Motor function performance was evaluated and the neuroinflammatory response and gene expression pattern in brain and spinal cord were studied by immunostaining and qRT-PCR. We found that VCE-003 ameliorated the symptoms associated to TMEV infection, decreased microglia reactivity and modulated the expression of genes involved in MS pathophysiology. These data lead us to consider VCE-003 to have high potential for drug development against MS and perhaps other neuroinflammatory diseases.

Synthesis and biological evaluation of 12-aminoacylphorboids.

Spurred by the paradoxical anti-inflammatory activity of some aminoacylphorbol derivatives, the naturally occurring and epimeric N,N-dimethylvalinoyl-4alpha-4-deoxyphorbol derivatives 3b and 3d have been prepared from 4alpha-4-deoxyphorbol (3e), a byproduct of the isolation of phorbol from Croton oil and a phorboid polyol so far largely overlooked in terms of biological activity. The configuration of the side chain stereocenter was confirmed for both natural products, and to investigate the side chain structure-activity relationships within this class of compounds, their corresponding N,N-dimethylglycinate (3g) and nor (3h) and di-nor derivatives (3i, 3j) were also prepared. By using a PKC-sensitive model of HIV-1 latency (activation of HIV- gene expression in Jurkat-LAT-GFP cells), it was found that both 3b and 3d can activate PKC-dependent responses, while a series of experiments with isoform-specific PKC inhibitors showed that these compounds target PKCalpha and -delta. Both N,N-dimethylation and the presence of side chain alpha-substitution were critical for activity. Selective PKC binding, rather than COX inhibition, might explain the paradoxical anti-inflammatory activity of extracts containing aminoacylphorboids in the mouse ear edema assay.

Modulation of the transient receptor potential vanilloid channel TRPV4 by 4alpha-phorbol esters: a structure-activity study.

The mechanism of activation of the transient receptor potential vanilloid 4 (TRPV4) channel by 4alpha-phorbol esters was investigated by combining information from chemical modification of 4alpha-phorbol-didecanoate (4alpha-PDD, 2a), site-directed mutagenesis, Ca(2+) imaging, and electrophysiology. Binding of 4alpha-phorbol esters occurs in a loop in the TM3-TM4 domain of TRPV4 that is analogous to the capsaicin binding site of TRPV1, and the ester decoration of ring C and the A,B ring junction are critical for activity. The lipophilic ester groups on ring C serve mainly as a steering element, affecting the orientation of the diterpenoid core into the ligand binding pocket, while the nature of the A,B ring junction plays an essential role in the Ca(2+)-dependence of the TRPV4 response. Taken together, our results show that 4alpha-phorbol is a useful template to investigate the molecular details of TRPV4 activation by small molecules and obtain information for the rational design of structurally simpler ligands for this ion channel.

Antibacterial cannabinoids from Cannabis sativa: a structure-activity study.

Marijuana (Cannabis sativa) has long been known to contain antibacterial cannabinoids, whose potential to address antibiotic resistance has not yet been investigated. All five major cannabinoids (cannabidiol (1b), cannabichromene (2), cannabigerol (3b), Delta (9)-tetrahydrocannabinol (4b), and cannabinol (5)) showed potent activity against a variety of methicillin-resistant Staphylococcus aureus (MRSA) strains of current clinical relevance. Activity was remarkably tolerant to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids), and to carboxylation of the resorcinyl moiety (pre-cannabinoids). Conversely, methylation and acetylation of the phenolic hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were all detrimental for antibacterial activity. Taken together, these observations suggest that the prenyl moiety of cannabinoids serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore, while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.

Differential effects of phorbol-13-monoesters on human immunodeficiency virus reactivation.

The persistence of latent reservoirs of HIV-1 represents a major barrier to virus eradication in patients treated with antiretrovirals. Prostratin is a non-tumor promoting 12-deoxyphorbol monoester capable of up-regulating viral expression from latent provirus and therefore is potentially useful for HIV adjuvant therapy and similar properties might be elicited by related non-tumor promoting phorboids. We have therefore investigated a series of phorbol 13-monoesters for their capacity to reactivate HIV latency. Using a Jurkat T cell line containing latent HIV proviruses, we found that prostratin and phorbol-13-stearate effectively activate HIV-1 gene expression in these latently infected cells, with phorbol-13-stearate being at least 10-fold more potent than prostratin, and its activity rapidly decreasing with a shortening of the acyl side chain. We further demonstrated that phorbol-13-stearate and prostratin stimulate IKK-dependent phosphorylation and degradation of IkappaBalpha, leading to activation of NF-kappaB. Moreover, prostratin, phorbol-13-hexanoate and phorbol-13-stearate also activate the JNK and ERK pathways. Studies with isoform-specific PKC inhibitors suggest that the classical PKCs play a prominent role in the responses elicited by phorbol-13-stearate. Nevertheless, this compound induces a translocation pattern of the PKC isotypes alpha and delta to cellular compartments distinctly different from that elicited by prostratin and PMA.

alpha-fetoprotein expression in a dedifferentiated liposarcoma.

Extremely rare cases of paraneoplastic syndromes or ectopic production of proteins associated with liposarcoma are reported in literature. We describe a unique case of relapsing retroperitoneal dedifferentiated liposarcoma with biochemical, immunohistochemical, and molecular evidence of alpha-fetoprotein (AFP) ectopic production. The lesion was associated to elevated AFP plasma levels that subsided after tumor removal. Immunohistochemical studies showed AFP production by a minority of tumor cells and reverse transcriptase polymerase chain reaction confirmed AFP mRNA expression. Finding of MDM2 and CDK4 iperexpression by immunohistochemistry confirmed the diagnosis of dedifferentiated liposarcoma.

Potential applications of molecular biology in neuroendocrine tumors.

The impact of molecular biology procedures on neuroendocrine (NE) tumor biology is gradually evolving from purely academic and research studies to clinical applications. This review deals with applications of molecular techniques in neuroendocrine tumors, with special reference to their potential for diagnostic, prognostic, or therapeutic impact. Since the cloning of the genes involved in inherited endocrine tumor syndromes, molecular analysis of the responsible genetic alterations has become a routine diagnostic tool to select affected patients and their relatives, and also an interesting approach to investigate the pathogenesis of neuroendocrine tumors. Assessment of the clonal composition of endocrine tumors could be useful to differentiate hyperplastic versus either adenomatous or carcinomatous conditions, as well as to better understand the clonal relationship between different neoplastic populations in mixed tumors. In addition, molecular approaches allow high sensitivity both in defining the neuroendocrine phenotype in poorly differentiated tumors and in searching for micrometastasis during the follow up of patients with endocrine tumors. Finally, the detection of peptide hormone receptors (e.g., oxytocin and somatostatin receptors) and the development of potent synthetic analogs of such peptides, are opening promising applications in the diagnosis and therapy of endocrine tumors.

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 10(3) to 10(6) (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

Primitive Neuroectodermal Tumor of the Meninges: An Histological, Immunohistochemical, Ultrastructural, and Cytogenetic Study.

We report a case of primitive neuroectodermal tumor (PNET) arising from the meninges in a 30-yr-old female patient whose clinical and radiological features were consistent with meningioma. The neoplasm was composed of primitive small, round cells, growing in sheets and nests in continuity with meningeal layers. Ultrastructurally, the neoplastic cells were characterized by large and regular nuclei, primitive cytoplasm with pools of glycogen, and lack of dense core granules. The neuroectodermal nature of the tumor was confirmed by positive immunostaining for vimentin, neurofilaments, neuron specific enolase (NSE), and 013 (an antibody raised against MIC2 antigen). Further support to the diagnosis was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) detection of Chromogranin A and Secretogranin II genes. t(1 1;22) translocation was also observed by RT-PCR, a finding that was not recorded in previously described intracranial PNET. The tumor followed a malignant course, recurring and spreading to chest wall and sacroiliac region over a 10-yr period. The meningeal location enlarges the topographic spectrum of intracranial PNET, and this tumor has to be considered in the differential diagnosis with meningioma.