A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an α-lactalbumin-oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured α-lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here.

HPV16 E5 is produced from an HPV16 early mRNA spliced from SD226 to SA3358.

The HPV16 E5 open reading frame (ORF) is present on the majority of all alternatively spliced HPV16 mRNAs, but it is currently unknown how well it is translated into E5 protein. To identify HPV16 mRNAs that are efficiently translated into E5, we have generated cDNA plasmids expressing individual, alternatively spliced HPV16 mRNAs with the potential to produce E5. By replacing the E5 ORF with sLuc, we could quantitate sLuc and determine how well each cDNA was translated. Our results showed that the upstream E1 and E7 AUGs inhibited translation of the E5 ORF and revealed that only one HPV16 mRNA produced high levels of E5. This was an HPV16 early mRNA spliced from SD226 to SA3358. These results were confirmed in the context of the entire HPV16 genome. Taken together, our results indicate that E5 is expressed early in the HPV16 replication cycle since it is translated efficiently only by one early mRNA.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3'-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Acetylation of intragenic histones on HPV16 correlates with enhanced HPV16 gene expression.

We report that many histone modifications are unevenly distributed over the HPV16 genome in cervical cancer cells as well as in HPV16-immortalized keratinocytes. For example, H3K36me3 and H3K9Ac that are common in highly expressed cellular genes and over exons, were more common in the early than in the late region of the HPV16 genome. In contrast, H3K9me3, H4K20me3, H2BK5me1 and H4K16Ac were more frequent in the HPV16 late region. Furthermore, a region encompassing the HPV16 early polyadenylation signal pAE displayed high levels of histone H3 acetylation. Histone deacetylase (HDAC) inhibitors caused a 2- to 8-fold induction of HPV16 early and late mRNAs in cervical cancer cells and in immortalized keratinocytes, while at the same time increasing the levels of acetylated histones in the cells and on the HPV16 genome specifically. We concluded that increased histone acetylation on the HPV16 genome correlates with increased HPV16 gene expression.

Suppression of HPV-16 late L1 5'-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs.

Human papillomavirus type 16 (HPV-16) 5'-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5'-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production.

HAMLET binding to α-actinin facilitates tumor cell detachment.

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in ß1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

BACKGROUND: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. METHODOLOGY/PRINCIPAL FINDINGS: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. CONCLUSIONS/SIGNIFICANCE: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes.

Human α-lactalbumin made lethal to tumor cells (HAMLET) and equine lysozyme with oleic acid (ELOA) are complexes consisting of protein and fatty acid that exhibit cytotoxic activities, drastically differing from the activity of their respective proteinaceous compounds. Since the discovery of HAMLET in the 1990s, a wealth of information has been accumulated, illuminating the structural, functional and therapeutic properties of protein complexes with oleic acid, which is summarized in this review. In vitro, both HAMLET and ELOA are produced by using ion-exchange columns preconditioned with oleic acid. However, the complex of human α-lactalbumin with oleic acid with the antitumor activity of HAMLET was found to be naturally present in the acidic fraction of human milk, where it was discovered by serendipity. Structural studies have shown that α-lactalbumin in HAMLET and lysozyme in ELOA are partially unfolded, 'molten-globule'-like, thereby rendering the complexes dynamic and in conformational exchange. HAMLET exists in the monomeric form, whereas ELOA mostly exists as oligomers and the fatty acid stoichiometry varies, with HAMLET holding an average of approximately five oleic acid molecules, whereas ELOA contains a considerably larger number (11- 48). Potent tumoricidal activity is found in both HAMLET and ELOA, and HAMLET has also shown strong potential as an antitumor drug in different in vivo animal models and clinical studies. The gain of new, beneficial function upon partial protein unfolding and fatty acid binding is a remarkable phenomenon, and may reflect a significant generic route of functional diversification of proteins via varying their conformational states and associated ligands.

HAMLET treatment delays bladder cancer development.

PURPOSE: HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. MATERIALS AND METHODS: Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. RESULTS: HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. CONCLUSIONS: Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development.

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

BACKGROUND: Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. METHODOLOGY/PRINCIPAL FINDINGS: We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. CONCLUSIONS/SIGNIFICANCE: The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

alpha-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-lactalbumin on the stationary phase in situ. Because native alpha-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

Histone deacetylase inhibitors promote the tumoricidal effect of HAMLET.

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.

Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

We studied if bladder cancers respond to HAMLET (human alpha-lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native alpha-lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.

alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.

Compact oleic acid in HAMLET.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex between alpha-lactalbumin and oleic acid that induces apoptosis in tumor cells, but not in healthy cells. Heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of 13C-oleic acid in HAMLET, and to study the 15N-labeled protein. Nuclear Overhauser enhancement spectroscopy shows that the two ends of the fatty acid are in close proximity and close to the double bond, indicating that the oleic acid is bound to HAMLET in a compact conformation. The data further show that HAMLET is a partly unfolded/molten globule-like complex under physiological conditions.

HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy.

New cancer treatments should aim to destroy tumor cells without disturbing normal tissue. HAMLET (human alpha-lactalbumin made lethal to tumor cells) offers a new molecular approach to solving this problem, because it induces apoptosis in tumor cells but leaves normal differentiated cells unaffected. After partial unfolding and binding to oleic acid, alpha-lactalbumin forms the HAMLET complex, which enters tumor cells and freezes their metabolic machinery. The cells proceed to fragment their DNA, and they disintegrate with apoptosis-like characteristics. HAMLET kills a wide range of malignant cells in vitro and maintains this activity in vivo in patients with skin papillomas. In addition, HAMLET has striking effects on human glioblastomas in a rat xenograft model. After convection-enhanced delivery, HAMLET diffuses throughout the brain, selectively killing tumor cells and controlling tumor progression without apparent tissue toxicity. HAMLET thus shows great promise as a new therapeutic with the advantage of selectivity for tumor cells and lack of toxicity.

Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex.

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.

Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid.

BACKGROUND: We studied the effect on skin papillomas of topical application of a complex of alpha-lactalbumin and oleic acid (often referred to as human alpha-lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that alpha-lactalbumin-oleic acid kills transformed cells but not healthy, differentiated cells. METHODS: Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which alpha-lactalbumin-oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of alpha-lactalbumin-oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term follow-up data were obtained. RESULTS: In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the alpha-lactalbumin-oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with alpha-lactalbumin-oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with alpha-lactalbumin-oleic acid, and the time to resolution was shorter in the group originally assigned to receive alpha-lactalbumin-oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients. CONCLUSIONS: Treatment with topical alpha-lactalbumin-oleic acid has a beneficial and lasting effect on skin papillomas.